Compositions and methods to control abnormal cell growth

ABSTRACT

A class of compounds commonly containing a trialkylammonium group have been synthesized and characterized as anticancer compounds. A representative of this class, N,N-diethyl-N-methyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]ethanaminium iodide (CCDTHT) was shown in various tumor models to decrease tumor volume, enhance the effects of other chemotherapeutic agents including cisplatin, reduce chemotherapy-induced loss of body weight, and increase survival of animals co-treated with toxic amounts of cisplatin. CCDTHT had even greater effects on tumor volume, body weight, and survival when administered to animals together with the human protein placental alkaline phosphatase. These trialkylammonium group-containing compounds and alkaline phosphatases, particularly in combination with each other and other therapies, may be used to treat cancer and other cell proliferative diseases.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional of U.S. application Ser. No.11/458,502, filed Jul. 19, 2006, which claims the benefit of U.S.Provisional Application No. 60/716,346, filed Sep. 12, 2005. Theseapplications are incorporated herein by reference.

FIELD OF THE INVENTION

Embodiments of this invention include the use of a specific class ofchemically synthesized compounds and alkaline phosphatase, separately orin combination with each other and other therapies, to selectivelydecrease the viability of non-healthy cells, particularly cancer cellsin tumors that lost growth control. Other embodiments of the inventionalso use these chemically synthesized compounds and alkaline phosphataseto enhance the efficacy of chemotherapy with simultaneous reduction ofside effects.

BACKGROUND

Over a lifetime there is a potential for the development of manydifferent types of proliferative diseases in solid tissues characterizedby the loss of cellular growth control, such as cancer, psoriasis, orkeloid tissue. The diseased tissues and organs are often characterizedby either higher than normal rate of proliferation of the affected cells(usually at the expense of the surrounding normal cells) or theinability to stop proliferating when so signaled by appropriatesignaling mechanisms often activated by differentiation-inducing agents.Since in unhealthy and healthy tissues expression of various genesusually differs only quantitatively but not qualitatively, it isextremely difficult to employ a particular kind of chemotherapy thatselectively destroys only the non-healthy tissue. This is one reasonthat only few effective monotherapies exist against any of thesediseases that would be relatively free of toxic side effects.

Therefore, in recent years various combination chemotherapies havebecome standard procedures to attack these proliferative diseases,particularly cancers. While combination therapies are usually moreeffective than treatments with single agents, they are often even moretoxic than monotherapies that usually act via a single mechanism. Thus,it would be desirable to develop new anti-proliferative agents that addto the effects of known chemotherapies and simultaneously decrease theusual side effects such as significant weight loss accompanied byfatigue. For that purpose, embodiments of the present invention providea class of chemically synthesized agents and alkaline phosphatase,particularly the placental type alkaline phosphatase, both alone or incombination.

SUMMARY OF THE INVENTION

Embodiments of this invention include simultaneously enhancing theefficacy and decreasing the toxicity of chemotherapy by single agentsand particularly their combinations. These agents are also suitable toenhance the efficacy of other therapies aimed at selectively suppressingthe growth of non-healthy cells and tissues characterized byuncontrolled growth.

Finally, other embodiments of the invention include a class ofchemically synthesized compounds that, even when used as single agents,exert strong anti-cancer effects, for example, against ovarian, breastcancer, and other types of cancer cells.

The first class of agents used in embodiments of the invention arechemically synthesized. They were designed to inhibit cellular cholinetransport across the cell membrane and alter the membrane potential ofmitochondria. Both of these effects of chemically synthesized compounds(hereinafter referred to as “CC compounds”) are directed at non-healthycells with some specificity. A third separate mechanism of action invivo, specifically accounting for the ability of CC compounds todecrease or prevent chemotherapy-induced reduction in body weight, isvery likely. Representatives of CC compounds are CCcompound3 (or CCDTHT)and CCcompound26, although the effects of several more of thesecompounds are presented in embodiments of the invention.

Tn one embodiment, the invention provides for preferential killing ofnon-healthy abnormally growing cells, in vitro, by CC compounds. Inother embodiments, the invention provides methods to employ CCcompound3or CCDTHT and other CC compounds in humans and other mammals to decreasethe growth of abnormally growing tissues (including tumor tissues),normalize body weight, and decrease the toxic side effects caused bychemotherapy or other therapies. CCcompound3, CCcompound26 and mostother CC compounds are soluble in water and are suitable for oralapplication in the form of tablets, gel capsules and the like. They alsocan be administered by one of the available systemic routes.

The second agent used in embodiments of the invention is placentalalkaline phosphatase (PALP), a member of the alkaline phosphatasefamily. In one embodiment, the invention provides methods to employ PALPand other alkaline phosphatases in humans and other mammals to enhancethe efficacy of various treatments aimed at decreasing the growth ofabnormally growing tumor tissues. The terms “PALP” and “alkalinephosphatase” are used interchangeably throughout the application. Inanother embodiment, PALP is used to normalize body weight and decreasethe toxic side effects caused by chemotherapy and other treatments.Alkaline phosphatases can be applied for therapy via injection using oneof the available systemic routes.

Embodiments of the invention also provide for the use of CCcompound3 andother CC compounds as well as PALP and other alkaline phosphatases incombination to more effectively enhance the effects of other therapiesto control tumor growth as well as restore body weight and decreasetherapy-induced toxic side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a picture of a gel separation, demonstrating that the PALPused for the animal experiments shown in Tables 6 to 10 was homogeneousor near homogeneous.

FIG. 2 is a digital image showing that unlike untreated human melanomaMEL-28 cells (A), MEL-28 cells treated with 100 μM CCDTHT (CCcompound3)for 72 hours in 12-well plates were all rounded up, indicating death viaone of the apoptotic pathways (B).

FIG. 3 is a digital image indicating that, compared to untreated humanbreast carcinoma MCF-7 cells (A), treatment of MCF-7 cells with 100 μMCCDTHT (CCcompound3) for 72 hours (in 12-well plates) induced strongalterations in the morphology consistent with the death of all cells(B).

FIG. 4 is an image illustrating that unlike untreated human breastcarcinoma T47D cells (A), T47D cells treated with 100 μM CCDTHT(CCcompound3) for 72 hours in 12-well plates were all rounded up,indicating death via one of the apoptotic pathways (B).

FIG. 5 compares the anti-cancer effects of 50-200 μM concentrations ofcommercially available CCcompound1 () as well as CCDTHT (CCcompound3)(▴) on the viability of NIH 3T3, MEL-28, A549, HT-29, A-431 and MCF-7cells after treatments for 72 hours.

FIG. 6 shows the effects of 50 μM and 100 μM CCDTHT on cell viability inthe normal HTB-157 and NIH 3T3 fibroblasts as well as 10 establishedcancer cell lines including T47D, ZR-75-1, MB-231, CaOV-3, A-431,MEL-24, MEL-28, HT-29, CaCO-2, and HepG2 cells after continuoustreatments for 10 days.

FIG. 7 shows the effects of 5, 100, 150 and 200 μM CCDTHT on theviability of U-87 and CCF-STT GI cancer cells after treatments for 72hours.

FIG. 8 depicts an experiment performed with six different types ofcells. HTB-157, MEL-28, 3A-SubE, CaOV-3, T47D and A-431 cells were firsttreated for 48 hours with 50 μM or 100 μM CCDTHT (48 h) and thenincubated in fresh medium (in the absence of CCDTHT) for 96 hours (48+96h); the relative numbers of viable cells are shown.

FIG. 9 shows that in the AN3CA MCF-7 and MEL-28 human cancer cells, 100μM CCDTHT

and 0.5 mM HC-3 (▪) exerted similarly strong inhibitory effects on thecellular uptake of [¹⁴C]choline (A) as well as on the synthesis of[¹⁴C]PCho (B) and [¹⁴C]PtdCho (C) from radiolabeled choline aftertreatments for 2 hours. The symbol “□” represents the correspondingvalues in the untreated cells.

FIG. 10 demonstrates that in AN3CA, HT-29 and MEL-28 cancer cells, butnot in MCF-7 cancer cells, 100 μM CCDTH

caused a much larger decrease in the number of viable cells than 0.5 mMHC-3 did (▪) after treatments for 72 hours. The symbol “□” representsthe corresponding viability values in the untreated cells.

FIG. 11 shows that in MCF-7 cells, but not in AN3CA, HT-29, or MEL-28cells, 2 mM ethanolamine (Etn) prevented the large decrease in viabilityinduced by 100 μM CCDTHT after treatments for 72 hours. In the NIH3T3non-cancerous cells CCDTHT had no significant effects either in theabsence or presence of Etn.

FIG. 12 shows that treatments of HaCaT keratinocytes with 50 μM or 100μM CCDTHT for 48 hours resulted in the death of 85-100% of cells.Similar treatments of HTB-157 fetal (normal) fibroblasts with CCDTHTcaused only 19-20% inhibition of cell proliferation.

FIG. 13 shows an experiment in which the effects of 50 μM and 100 μMCCDTHT were compared on the viability of HaCaT cells as well as 966 SK,HTB-157, and NIH 3T3 normal fibroblasts after treatments for 72 hours.

FIG. 14 shows that in human leukemia HL60 cells, human melanoma HT-168cells, human prostate cancer PC-3 cells and human breast cancer T47Dcells 25 μM CCDTHT inhibited cell proliferation by 40-55%, while 75 μMCCDTHT decreased the number of viable cells by ˜65-85% if treatmentswere for 96 hours.

FIG. 15 depicts the effects of 50 μmole

, 100 μmole

, 200 μmole

, and 400 μmole CCDTHT (▪), injected subcutaneously, on the growth ofHL-60 (leukemia), HT-169 (melanoma), PC-3 (prostate), and T47D (breast)human tumor xenografts. The symbol “□” indicates the correspondingvalues in the untreated cells. In a, b, c, d treatments started on day18, 17, 12 and 17, respectively; once daily treatments were performedfor 5+3 days, 5+5 days, 5+3 days and 5+5 days, respectively, in eachcase with two treatment-free days between the two treatment series. Thevalues represent tumor size measured one day after the last treatment.

FIG. 16 demonstrates the growth of human melanoma HT-168 xenograftsbetween 17-30 days with no treatment (), or with once daily treatmentson days 17, 18, 19, 20, 21, 24, 25, 26, 27 and 28 with 200 μmole CCDTHT(▴), 100 μmole pyrrolidinedithiocarbamate (PC)+100 μmole zinc chloride(Zn) (▪), or 200 μmole CCDTHT+100 μmole PC+100 μmole Zn (♦).

DETAILED DESCRIPTION OF THE INVENTION

Mitochondria in cancer cells are characterized by significantly highermembrane potential than normal cells due to a high negative charge.Positively charged compounds can pass through the hydrophobic barrier ofa cell (plasma) membrane, particularly if they are sufficientlyhydrophobic, but they are retained in the mitochondrial membrane due tothe negative charge of the latter. This can affect the function ofmitochondria and lead, eventually, to cell dead. A well establishedprototype of such a positively charged compound is rhodacyanine MKT 077or1-ethyl-2-{[3-ethyl-5-(3-methylbenzothiazolin-2-yliden]-4-oxothiazolidin-2-ylidenemethyl}pyridium chloride [Kawakami, M., Koya, K., Ukai, T., Tatsuta, N.,Ikegawa, A., Ogawa, K., Shishido, T. and Chen, L. B. (1998),“Structure-activity of novel rhodacyanine dyes as antitumor agents,” J.Med. Chem. 41, 130-142; Chiba, Y., Kubota, T., Watanabe, M., Otani, Y.,Teramoto, T., Matsumoto, Y., Koya, K. and Kitajima, M. (1998), Selectiveantitumor activity of MKT-077, a delocalized lipophilic cation, onnormal cells and cancer cells in vitro,” J. Surgical Oncol. 69,105-110]. Unfortunately, phase I human trials revealed that MKT 077exerts significant renal toxicity which prevents its use in humansubjects [Propper, D. J., Braybrooke, J. P., Taylor, D. J., Lodi, R.,Styles, P., Cramer, J. A., Collins, W. C. J., Levitt, N. C., Talbot, D.C., Ganesan, T. S. and Harris, A. L. (1999), “Phase I trial of theselective mitochondrial toxin MKT 077 in chemo-resistant solid tumours,:Annals of Oncol. 10, 923-927; Britten, C. D., Rowinsky, E. K., Baker, S.D., Weiss, G. R., Smith, L., Stephenson, J., Rothenberg, M., Smetzer,L., Cramer, J., Collins, W., Von Hoff, D. D. and Eckhardt, S. G. (2000),“A phase I and pharmacokinetic study of the mitochondrial-specificrhodacyanine dye analog MKT 077,“Clin. Cancer Res. 6, 42-49]. Evidently,a positively charged compound which has the potential to interact withand decrease the membrane potential of mitochondria would be expected toeffectively inhibit proliferation of cells with some specificity towardcancer cells. In embodiments of this invention, various molecules (“CCcompounds”) that have both hydrophobic and hydrophilic moieties with anet positive charge may kill abnormal cells, including cancer cells, invitro and in vivo.

Recent research has revealed that cancer cells exhibit high cholinekinase activity, an enzyme that produces phosphorylcholine (PCho, alsoknown as choline phosphate) from the choline and adenosinetriphosphateprecursors. For that reason, direct inhibitors of choline kinase exhibitanti-cancer activity in various in vivo tumor models[Hernandez-Alcoceba, R., Fernandez, F. and Lacal, J. C. (1999), “In vivoantitumor activity of choline kinase inhibitors: A novel target foranticancer drug discovery,” Cancer Res. 59, 3112-3118; de Molina, A. R.,Gutierrez, R., Ramos, M. A., Silva, J. M., Silva, J., Bonilla, F.,Sanchez, J. S. and Lacal, J. C. (2002), “Increased choline kinaseactivity in human breast carcinomas: clinical evidence for a potentialnovel antitumor strategy,” Oncogene 21, 4317-4322]. Another possiblemechanism to decrease the synthesis of PCho is via inhibiting cholineuptake system in the target cells. Rapidly proliferating cells, such asmost cancer cells, have higher choline transport capacity than normalcells to satisfy the need of phosphatidylcholine (PtdCho) biosynthesisfor this precursor. PtdCho is a major phospholipid that is an essentialbuilding block of biological membranes. Therefore, any disruption ofPtdCho synthesis will have a negative impact on cell proliferation.Accordingly, inhibitors of choline transport are expected topreferentially inhibit rapid proliferation of non-healthy cells in vivowith less impact on normal tissues. Based on these considerations, theCC compounds were designed to contain moieties that can interfere withcellular uptake of choline from the extracellular milieu in addition toreducing membrane potential in the mitochondria.

There are two different transport systems for choline. Thesodium-dependent, high-affinity transport system is specificallylocalized to cholinergic nerve terminals and provides choline foracetylcholine synthesis. The low-affinity choline transport system ispresent in most cell types, including those dealt with in embodiments ofthe present invention, and provides choline for the synthesis of PChoand PtdCho [Slack, B. E., Breu, J., Livneh, E., Eldar, H. and Wurtman,R. J. (1995), “Phorbol ester stimulates choline uptake in Swiss 3T3fibroblasts following introduction of the gene encoding protein kinaseCα, Biochem. J. 305, 621-626; and references therein]. Hemicholinium-3,or HC-3 [2,2′-(4,4′-biphenylene)-bis(2-hydroxy-4,4-dimethylmorpholiniumbromide], is a more effective inhibitor of the high-affinity cholinetransport system, compared to the low-affinity system, resulting in thedepletion of acetylcholine stores. For this reason, HC-3 and somerelated bis-hemiketal compounds are toxic causing delayed andprogressive respiratory failure [Happe, H. K. and Murrin, L. C. (1993),“High-affinity choline transport sites: Use of [³H]hemicholinium-3 as aquantitative marker,” J. Neurochem. 60, 1191-1201; Cannon, J. G. (1994),“Structure-activity aspects of hemicholinium-3 (HC-3) and its analogsand congeners,” Medicinal Res. Rev. 14, 505-531]. Clearly, forinhibiting PCho and PtdCho synthesis in rapidly growing aberrant cells,it would be necessary to use a choline transport inhibitor that, unlikeHC-3, is non-neurotoxic (or significantly less toxic); i.e. thatpreferentially inhibits the low-affinity transport system. CC compoundsin embodiments of the present invention are sufficiently different fromHC-3 to have less toxicity, but they also contain a group that has theability to compete with choline for the low affinity transport system.Because CC compounds contain a positive charge (due to the quaternaryammonium), these compounds also only poorly penetrate, if at all, thebrain-blood barrier that further decreases their potential inhibitoryeffects on neurotransmission.

The choline transport system in rapidly proliferating cells,particularly cancer cells, is more abundant than in normal cells. Sincethe concentration of choline in the blood is only around 25 μM, therapidly growing tumor tissue will use most of the choline with littlecholine left for the normal cells. However, since in most normalestablished tissues only a very low level of cell proliferation is goingon, these tissues can survive and function normally even at very lowblood choline levels. Thus, it was expected that concentrations of CCcompounds that inhibit tumor growth via inhibiting choline uptake shouldhave no or much less toxic effects in normal tissues.

It was reported earlier that placental alkaline phosphatase (PALP), oneof the presently known four members of the alkaline phosphatase enzymefamily [J. L. Millan and W. H. Fishman (1995), “Biology of humanalkaline phosphatases with special reference to cancer,” CriticalReviews in Clinical Sciences 32, 1-39], can enhance both theproliferation and survival of mouse embryo fibroblasts as well asfibroblast-like cells derived from the lung of human fetus [Q. -B. She,J. J. Mukherjee, J. -S. Huang, K. S. Crilly, and Z. Kiss (2000), “Growthfactor-like effects of placental alkaline phosphatase in human fetus andmouse embryo fibroblasts,” FEBS Letters, 468, 163-167; Q. -B. She, J. J.Mukheijee, T. Chung, and Z. Kiss (2000), “Placental alkalinephosphatase, insulin, and adenine nucleotides or adenosinesynergistically promote long-term survival of serum-starved mouse embryoand human fetus fibroblasts,” Cellular Signalling 12, 659-665]. In tworecent U.S. patent applications filed by the present inventor, PALP wasshown to also enhance proliferation of human fibroblasts [U.S. Pat. No.7,011,965, entitled “Compositions and Methods for Stimulating WoundHealing and Fibroblast Proliferation”; U.S. patent application Ser. No.10/653,622, filed Sep. 2, 2003 and entitled “Use of Placental AlkalinePhosphatase to Promote Skin Cell Proliferation”]. In both patentapplications, the effects of PALP alone on wound healing and skin careapplications are reported.

In the present application, PALP is used either alone or in combinationwith a CCcompound and/or other chemotherapeutic agents to decrease tumorgrowth and restore chemotherapy-induced loss of body weight. For this,PALP was highly purified from commercial (Sigma-Aldrich) PALP preparedby a previously described method with minor modifications [Q.-B. She, J.J. Mukherjee, J. -S. Huang, K. S. Crilly, and Z. Kiss (2000), “Growthfactor-like effects of placental alkaline phosphatase in human fetus andmouse embryo fibroblasts,” FEBS Letters, 468, 163-167]. Analysis by gelelectrophoresis and peptide analysis shows that in the PALP preparationspurchased from Sigma-Aldrich, PALP represents about 10% of the totalprotein, the remaining being represented by α₁-antitrypsin, transferrin,albumin, and few other lower molecular mass contaminating proteinsmostly derived from transferrin.

The alkaline phosphatase family also includes the tissue non-specific(liver/bone/kidney) alkaline phosphatase, the intestinal alkalinephosphatase, and the PALP-like (germ-cell) alkaline phosphatase. Sinceeach of these enzymes has similar phosphatase activities that maycontribute to the negative control of cancer cell growth, these threeenzymes may share, at least partially, the anti-cancer effects of PALP.

Amifostine is one of the few non-growth factor agents that may providesome protection against the toxic effects of chemotherapy in specificcases [for example, Verstappen, C. C. P., Postma, T. J., Geldof, A. A.and Heimans, J. J. (2004), “Amifostine protects againstchemotherapy-induced neurotoxicity: An in vitro investigation,”Anticancer Res. 24, 2337-2342; Kemp, G., Rose, P., Lurain, J., Berman,M., Manetta, A., Roullet, B., Homesley, H., Belpomme, D. and Glick, J.(1996), “Amifostine pretreatment for protection againstcyclophosphamide-induced and cisplatin-induced toxicities: Results of arandomized control trial in patients with advanced ovarian cancer,” J.Clin. Oncol. 14, 2101-2112] and radiation therapy [Brizel, D. M.,Wasserman, T. H., Henke, M., Strnad, V., Rudat, V., Monnier, A.,Eschwege, F., Zhang, J., Russell, L., Oster, W. and Sauer, R. (2000),“Phase III randomized trial of amifostine as a radioprotector in headand neck cancer,” J. Clin. Oncol. 18, 3339-3345]. Apart from the factthat amifostine often causes side effects such as nausea, vomiting,hypotension and allergic reaction, an even more serious concern is thatit may actually enhance the survival of tumor cells in vivo [Verstappen,C. C. P., Postma, T. J., Geldof, A. A. and Heimans, J. J. (2004),“Amifostine protects against chemotherapy-induced neurotoxicity: An invitro investigation,” Anticancer Res. 24, 2337-2342]. Other agents thatmay provide some protection against specific chemotherapeutic agentsinclude the flavonoid Frederine [van Acker, F. A. A., Boven, E., Kramer,K., Haenen, G. R. M. M., Bast, A. and van der Vijgh, W. J. F. (2001),“Frederine, a new and promising protector against doxorubicin-inducedcardiotoxicity,” Clin. Cancer Res. 7, 1378-1384], glutamine [Vahdat, L.,Papadopoulos, K., Lange, D., Leuin, S., Kaufinan, E., Donovan, D.,Frederick, D., Bagiella, E., Tiersten, A., Nichols, G., Garrett, T.,Savage, D., Antman, K., Hesdoffer, C. S. and Balmaceda, C. (2001),“Reduction of paclitaxel-induced peripheral neuropathy with glutamine,”Clin. Cancer Res. 7, 1192-1197], and salicylate [Li, G., Sha, S. -H.,Zotova, E., Arezzo, J., van de Water, T. and Schacht, J. (2002),“Salicylate protects hearing and kidney function from cisplatin toxicitywithout compromising its oncolytic action,” Lab. Invest. 82, 585-596].

Among the known growth-regulatory agents, Granulocyte colony stimulatingfactor, interleukin-6, and erythropoietin or Darbapoetin alfa act tonormalize blood cell counts which then helps to overcomechemotherapy-related or other cancer-therapy-related fatigue.Unfortunately, all these agents not only work with limited efficacy,they also exert certain side effects on their own. Furthermore, none ofthese agents have demonstrated positive effects on the life expectancyof cancer patients. Finally, a recent report suggests thaterythropoietin may actually impair, not improve, survival of cancerpatients [Brower, V. (2003), “Erythropoietin may impair, not improve,cancer survival,” Nature Med. 9, 1429, and references therein].

Based on these data in the literature, it is clear that there is anunmet need for agents or combinations of agents that can simultaneouslyenhance the efficacy and tolerability of various forms of cancer therapyincluding chemotherapy and radiotherapy. Embodiments of the presentinvention provide selected CC compounds and PALP as well as theircombinations that increase the effects of chemotherapy on tumor size,prevent chemotherapy-induced loss of body weight, and extend thesurvival of experimental animals in some tumor models. These CCcompounds and alkaline phosphatase may also have positive effects whencombined with other forms of cancer therapy such as radiotherapy andsurgery.

Active Compositions

Embodiments of the present invention provide chemically synthesizedcompounds that contain one or two quaternary ammonium group(s) attachedto a non-phospholipid and non-phosphorous heterocyclic hydrophobicmoiety to reduce the viability of rapidly proliferating abnormal cells.In this group of compounds, three alkanes, alkenes, alcohols or amines,or their combinations, replace three hydrogen atoms in the ammoniummoiety. These compounds were designed to enter the cell interior andinteract with the mitochondrial membrane as well as inhibit cholinetransport and thereby the synthesis of PCho and PtdCho. Since rapidlyproliferating cells require proportionally more PtdCho, inhibitors ofcholine transport are expected to more effectively inhibit the growth ofthe hyperproliferating abnormal cells, such as psoriatic keratinocytesand malignant cells, than that of normal cells.

A general formula to represent the members of this class of compounds isas follows:

In this formula, R₁ and R₃₋₈ may be independently chosen from hydrogenor from C₁-C₂₆ straight, branched or cyclic alkanes or alkenes, aromatichydrocarbons, alcohols, ethers, aldehydes, ketones, carboxylic acids,amines, amides, nitriles or five- and/or six-membered heterocycle aswell as their derivatives.

Further, the variables, R₉ and R₁₀, considered or taken together may be═O or ═CH-L-N⁺(R₁₁, R₁₂, R₁₃) (where -L-N⁺(R₁₁, R₁₂, R₁₃) is definedbelow). In addition, R₉ and R₁₀ considered or taken independently may be—OH or -L-N⁺(R₁₁, R₁₂, R₁₃).

The variables, V and Y, may be —S— or —Se—. Alternatively, the sulfurand selenium atoms in the heterocyclic moiety may further be replacedwith the carbon, oxygen, or silicon atoms. In yet other embodiments,either V or Y, or both, may be the N atom. In that case the -L-N⁺(R₁₁,R₁₂, R₁₃) group can also be attached to the N atom. For example, inphenoxazine the Y is N, and in that case the -L-N⁺(R₁₁, R₁₂, R₁₃) groupcan be linked to the N atom. Or in phenazine, both V and Y is N, and the-L-N⁺(R₁₁, R₁₂, R₁₃) group can be linked to both N atoms.

In the general formula, Z⁻ may be Cl⁻, Br⁻ or I⁻.

Also, in this general formula the variable, R₂, may be represented bythe following additional formula:

-X-L-N⁺(R₁₁, R₁₂, R₁₃)Z³¹

In this additional formula, X may be —CH₂—, —OCH₂—, —CH₂O—, —SCH₂— or—CH₂S—. Further, L may be C₁-C₄ straight alkane, alkene, thiol, ether,or amine.

The variables R₁₁, R₁₂ and R₁₃ may be represented by C₁-C₄ straightalkanes, alkenes, ethers, thiols, amines or alcohols. Preferably, theR₁₁, R₁₂, and R₁₃ groups are represented by methyl, ethyl, allyl,sulfhydryl, ether, amino, or hydroxyl groups or by their combinations.If one R₁₁, R₁₂ or R₁₃ group is an alcohol or an amine, this allowsfurther modification by targeting moieties (see below).

The above described quaternary ammonium-containing compounds may befurther altered to contain in alcohol or amine-containing X-L-N⁺(R₁₁,R₁₂, R₁₃) or ═CH-L-N⁺(R₁₁, R₁₂, R₁₃) groups a specific targeting moietyto direct the compound to specific tissues. Such targeting molecules maybe, for example, specific antibodies (recognizing antigens on thesurface of non-healthy cells), folic acid (recognizing highly expressedfolate receptor on the surface of certain types of tumor cells),steroids (recognizing the estrogen receptor in breast cancers), fattyacids, or specific peptides, such as transferrin, that can bind to cellsurface receptors. These targeting molecules as well as theircombination with alcohols or amines are well known to one havingordinary skill in this art.

Using an appropriate heterocyclic compound, such as for example4,7-phenanthroline or phenazine that has N atoms both in the 9th and10th positions, L-N⁺(R₁₁, R₁₂, R₁₃) moieties of varying compositions canbe attached to both N atoms. For example, in the first L-N⁺(R₁₁, R₁₂,R₁₃) moiety one of the R₁₁₋₁₃ groups may be an alcohol or amine whichallows further addition of a targeting group, while in the secondL-N⁺(R₁₁, R₁₂, R₁₃) moiety there would be no alcohol or amine.

In experiments performed in embodiments of the invention, certaincompounds described by the general formula were less effectiveinhibitors of choline transport and cancer cell proliferation. Forexample, several compounds, including celestine blue, gallocyanine,meldola blue, methylene blue, methylene green, methyl green and pyronin,all containing at least one quaternary nitrogen ion (with two methylgroups) attached via a double bond to a heterocyclic moiety, wereineffective or less effective than, for example, CCDTHT in inducingcancer cell death. Despite these observations, for the purpose ofembodiments of the invention, the thioxanthene or thioxanthone moietymay be replaced with any other heterocyclic moiety to which it isfeasible to attach at least one quaternary nitrogen group with threesubstituents that together are capable of inhibiting the transport ofexternal choline into cells and thereby inhibit cell proliferation.

Several subsets of thioxanthene/thioxanthone-based compounds weresynthesized in certain embodiments of the invention. Examples of thesynthesized compounds include, but are not limited to (i)N,N,N-trialkyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]ethanaminiumiodide, ii)N,N,N-trialkyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]propan-1-aminiumiodide and iii)N,N,N-trialkyl-3-(9H-thioxanthen-9-ylidene)-propan-1-aminium iodide.

One embodiment of these compounds isN,N-diethyl-N-methyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]-ethanaminiumiodide or CCDTHT (or CCcompound3), the structure of which is shown alongthe structure of other CCcompounds in Table 1. CCDTHT and all the otherCC compounds shown in Table 1, with the exception of CCcompound1, havebeen newly synthesized as reported the first time in this application,and are not available commercially. The synthesis of CCDTHT is describedin Example 1. The synthesis of CCcompounds17-20 as well as compounds 23,25 and 26 are also described in Examples 2-6. While CCDTHT was notalways the most effective CC compound in inhibiting the proliferation ofvarious cancer cells in vitro, it was selected for animal experimentsbased on its relatively low toxicity against normal cells in earlierexperiments. However, based on in vitro experiments with breast cancercells, it is expected that in some tumors, such as estrogenreceptor-positive breast cancer, CCcompound26 is more effective.

Single treatments with 450 μmole (about 4.6 mg/kg) CCDTHT or dailytreatments for 5 consecutive days with 400 μmole of CCDTHT did not causeany significant change in the composition of various blood constituentsin mice. Similarly, such treatments did not induce significantpathological alterations in the liver, brain, kidney, heart, spleen,intestine, and lung. However, significant alterations were observed inseveral tissues, accompanied by the death of 40% of animals by day 30,at the 900 μmole (about 9.2 mg/kg) dose of CCDTHT. Namely, 900 μmoleCCDTHT caused mild parenchymal degeneration in the heart, focalhypostasis and chronic bronchopneumonia in the lung, and multifocaldegeneration of hepatocytes in the liver. Accordingly, a well-tolerateddose for CCDTHT in mice is 450 μmole/animal (weighing about 25 g) whichcorresponds to a dose of 4.6 mg/kg. Therapeutically effectiveconcentrations in humans will exert maximal inhibitory effects on thegrowth of non-healthy tissues without causing significant toxicity innormal healthy tissues.

Parallel toxicological studies also have been performed with 200-500μmole/animal doses of the commercially available CCcompound1. It wassimilarly well tolerated by the treated mice, although in studiesperformed in vitro CCDTHT had less inhibitory effects on theproliferation of normal human fibroblasts compared to CCcompound1. Sincethe in vitro studies suggested that in the long-term CCcompound1 maydevelop some toxic side effects, most in vivo studies with mouse cancermodels were performed with CCDTHT. CCcompound26, which may have an evenbetter toxicity profile based on in vitro experiments with normal cells,was synthesized much later and has not yet been tested in animalexperiments.

TABLE 1 A Representative List of CC compounds Used in the Invention.Trivial name Chemical name Structure CCcompound 1[3-(3,4-Dimethyl-9-oxo- 9H-thioxanthen-2-yloxy)-2-hydroxypropyl]trimethyl- ammonium chloride

CCcompound 2 N,N,N-Trimethyl-2-[(9- oxo-9H-thioxanthen-2- yl)methoxy]-ethanaminium iodide

CCcompound 3 N,N-Diethyl-N-methyl-2- [9-oxo-9H-thioxanthen-2-yl)methoxy]- ethanaminium iodide

CCcompound 4 N,N,N-Triethyl-2-[(9-oxo- 9H-thioxanthen-2- yl)methoxy]-ethanaminium iodide

CCcompound 5 N-Ethyl-N,N-dimethyl-2- [(9-oxo-9H-thioxanthen-2-yl)methoxy]- ethanaminium iodide

CCcompound 6 2-{[2-(Diethylamino) ethoxy]methyl}-9H- thioxanthen-9-onehydrochloride

CCcompound 7 N,N,N-Trimethyl-3-[(9- oxo-9H-thioxanthen-2-yl)methoxy]-propane-1- aminium iodide

CCcompound 8 2-{[3-(Dimethylamino) propoxy]methyl}-9H- thioxanthen-9-onehydrochloride

CCcompound 9 N,N,N-Triethyl-3-[(9-oxo- 9H-thioxanthen-2-yl)methoxy]-propane-1- aminium iodide

CCcompound 10 N,N-Diethyl-N-methyl-3- [(9-oxo-9H-thioxanthen-2-yl)methoxy]-propane- 1-aminium iodide

CCcompound 11 N,N-Dimethyl-N-ethyl-3- [(9-oxo-9H-thioxanthen-2-yl)methoxy]-propane- 1-aminium iodide

CCcompound 12 2-{[3-(Diethylamino) propoxy]methyl}-9H- thioxanthen-9-onehydrochloride

CCcompound 13 2-Hydroxy-N,N- dimethyl-N-[(9-oxo-9H-thioxanthen-2-yl)methyl]- ethanaminium bromide

CCcompound 14 2-Hydroxy-N,N-Diethyl- N-[(9-oxo-9H-thioxanthen-2-yl)methyl]- ethanaminium bromide

CCcompound 15 3-Hydroxy-N,N-dimethyl- N-[(9-oxo-9H- thioxanthen-2-yl)methyl]propane-1- aminium bromide

CCcompound 16 3-Hydroxy-N,N-diethyl- N-[(9-oxo-9H-thioxanthen-2-yl)methyl]- propane-1-aminium bromide

CCcompound 17 3-(9-hydroxy-9H- thioxanthen-9-yl)-N,N,N-trimethyl-propane-1- aminium iodide

CCcompound 18 3-(9-hydroxy-9H- selenoxanthen-9-yl)-N,N,N-trimethyl-propane- 1-aminium iodide

CCcompound 19 N,N,N-trimethyl-3-(9H- thioxanthen-9-ylidene)-propane-1-aminium iodide

CCcompound 20 N,N,N-trimethyl-3-(9H- selenoxanthen-9-ylidene)-propane-1-aminium iodide

CCcompound 21 N,N,N-trimethyl-3-(2- methyl-9H-thioxanthen-9-ylidene)-propane-1- aminium iodide

CCcompound 22 N,N-Dimethyl-N-ethyl-3- (2-methyl-9H-thioxanthen-9-ylidene)- propane-1-aminium iodide

CCcompound 23 N,N-Diethyl-N-methyl-3- (2-methyl-9H-thioxanthen-9-ylidene)- propane-1-aminium iodide

CCcompound 24 N,N-Dimethyl-N-allyl-3- (2-methyl-9H-thioxanthen-9-ylidene)- propane-1-aminium bromide

CCcompound 25 N,N,N-Triethyl-3-(2- methyl-9H-thioxanthen-9-ylidene)-propane-1- aminium iodide

CCcompound 26 N,N-Diethyl-N-allyl-3-(2- methyl-9H-thioxanthen-9-ylidene)-propane-1- aminium bromide

The second agent used without or with CC compounds in the methods andcompositions in embodiments of the present invention is human placentalalkaline phosphatase (PALP), or an active derivative thereof.

As used herein, the term “PALP” and the phrase “human PALP” are usedinterchangeably to refer to placental alkaline phosphatase. The phrase“active PALP” means the human protein and its glycosylated andnon-glycosylated forms as well as peptides derived from these proteinsthat, when administered with or without CCcompounds and/or otherchemotherapeutic agents can increase the effectiveness of chemotherapyresulting in decreased tumor size, normalized body weight, and increasedlife expectancy.

PALP is a member of the alkaline phosphatase group of enzymes thathydrolyze phosphate-containing compounds at alkaline pH. Mature PALP isa dimer of two identical glycosylated subunits. Each subunit has anapproximate molecular weight of 66 kDa, as determined by gelelectrophoresis. Other members of this phosphatase group include thetissue non-specific (liver/bone/kidney), the intestinal, and PALP-like(germ-cell) alkaline phosphatases.

As mentioned earlier, PALP was found to enhance proliferation andsurvival of fibroblasts and some other types of healthy cells.Furthermore, it was demonstrated that PALP in its native stateexhibiting alkaline phosphatase activity is not required to achieve abeneficial effect on mitogenesis. For example, both digestion of PALPwith the protease bromelain and elimination of alkaline phosphataseactivity through mutation provided an active derivative [U.S. patentapplication Ser. No. 10/653,622, filed Sep. 2, 2003 and entitled “Use ofPlacental Alkaline Phosphatase to Promote Skin Cell Proliferation”; Pub.No. US2005/0048046 A1, Pub. Date, Mar. 3, 2005]. Consequently, one whois skilled in the art may synthesize or develop an active derivativethat is a smaller fragment of a PALP amino acid sequence anddemonstrates efficacy similar to that of native PALP. By way of example,modification of a PALP amino acid sequence or a sequence of smaller PALPpeptides by exchanging amino acids at critical sites to yield an activederivative may improve the beneficial effects of PALP in combinationwith the other proteins disclosed herein. Likewise, chemical orenzymatic changes in the level and position of glycosylation maymaintain or enhance the effects of PALP or its derivatives. In thepractice of embodiments of the present invention, it is envisioned thatmodified PALP, smaller PALP-derived peptides, or modified PALP-derivedpeptides may be similarly effective or even more effective than thenative PALP enzyme, and are each considered to be active derivatives.Likewise, PALP molecules isolated from placenta tissue or produced inrecombinant form are considered to be similarly effective.

While the stimulatory effects of PALP on the proliferation of normalcells may not require its enzyme activity, the effects of PALP on thesurvival and proliferation of cells in vivo may be, at least in part,the result of an indirect action. For example, PALP may activate immunecells via its phosphatase activity resulting in the release of cytokinesand growth factors that then may partly mediate the effects of PALP onnormal cells. Similarly, the major anti-cancer effects (for example,decrease of tumor size) of PALP may require phosphatase activity.Another possibility is that while phosphatase activity may not berequired for either effect, the structures of various alkalinephosphatases are sufficiently similar to cause similar effects on cellsurvival and proliferation. For example, it is known that all alkalinephosphatases interact in the plasma membrane with a common binding oranchoring site, called GPI anchor [J. L. Millan and W. H. Fishman(1995), “Biology of human alkaline phosphatases with special referenceto cancer.” Critical Reviews in Clinical Sciences 32, 1-39].Accordingly, each human alkaline phosphatase enzyme may at leastpartially mimic the effects of PALP both on normal as well as cancer andother abnormally growing cells.

Human PALP in solid form is available commercially from Sigma-Aldrich(St. Louis, Mo.), for example (Sigma catalog number P3895; CAS RegistryNumber 9001-78-9). Another commercial source of human PALP is Calbiochem(San Diego, Calif.; catalog number 524604).

Human PALP, and particularly a smaller molecular mass active derivative,may also be obtained by chemical synthesis using conventional methods.For example, solid-phase synthesis techniques may be used to obtain PALPor an active derivative.

Recombinant methods to obtain quantities of PALP (and active derivative)are also suitable. Since the cDNA of PALP is available, recombinantprotein can be produced by one of the many existing conventional methodsfor recombinant protein expression. PALP has been cloned andoverexpressed in a mammalian cell line [Kozlenkov, A., Manes, T.,Hoylaerts, M. F. and Millan, J. L. (2002), Function assignment toconserved residues in mammalian alkaline phosphatases,” J. Biol. Chem.277, 22992-22999; J. L. Millan and W. H. Fishman (1995), “Biology ofhuman alkaline phosphatases with special reference to cancer,” CriticalReviews in Clinical Sciences 32, 1-39]. Production of recombinant PALPby both bacteria [Beck, R. and Burtscher, H. (1994), “Expression ofhuman placental alkaline phosphatase in Escherichia coli,” ProteinExpression and Purification 5, 192-197] and yeast [Heimo, H., Palmu, K.and Suominen, I. (1998), Human placenta alkaline phosphatase: Expressionin Pichia pastoris, purification and characterization of the enzyme,”Protein Expression and Purification 12, 85-92] have also been reported.

Bacterial expression yields non-glycosylated PALP. So far there is noevidence that the anti-cancer effects of native glycosylated PALP andbacteria-produced PALP would be significantly different. Thus, in themethods of embodiments of the present invention native glycosylated PALPand its active derivatives as well as non-glycosylated PALP and itsactive derivatives may be used interchangeably.

A PALP preparation that is commercially available contains impurities.Impure PALP preparations can be used as starting material to obtainhomogeneous PALP by successive chromatographic steps, as described indetail in Example 1. Impure PALP preparations may also be used informulating the compositions for use in the practice of embodiments ofthe present invention, so long as the given composition comprisestherapeutically effective amount of PALP, and impurities are not toxicand do not interfere with the beneficial effects of the components.

A preparation containing human PALP may also be obtained by extractionfrom placental tissue. Human placenta synthesizes the enzyme duringpregnancy, so that toward the end of the third term, the level of PALPin the placenta tissue and the maternal/fetal blood becomes very high.By way of example, a preparation may be obtained by butanol extractionof homogenized placenta. Other methods of extraction from placentaltissue are also suitable.

Raw placental extracts that are not further enriched in PALP by usingphysical concentration methods cannot be expected to have physiologicaleffects similar to those observed for the preparation of sufficientlyenriched or purified or homogenous PALP, for at least two reasons.First, the relative concentration of PALP in an extract will be too lowto expect a readily detectable anti-cancer effect. Second, raw placentalextracts contain not only many different proteins but also other kindsof compounds, such as many lipids, proteolipids, carbohydrates, metals,vitamins, and the like that may cause unexpected side effects. Anadditional consideration is that only sufficiently highly purified PALPcan be introduced into the clinical practice to ensure the standardquality of the preparations and to exclude the health risks caused byunidentified contaminants.

Therefore, if placenta-derived PALP preparation is to be used in thepractice of embodiments of the present invention, a raw extract shouldbe treated to enrich the concentration of PALP and obtain asubstantially purified or highly purified preparation. A highly purifiedpreparation will have a much higher concentration of the activecomponent than found in a raw tissue extract. A highly purified PALPpreparation does not contain detectable amounts of contaminants orcontains such a minimum amount of known contaminants that the benefitsof using the preparation far out-weight the accompanying potentialrisks. The term “substantially purified” is used herein to encompasscompositions that are obtained from a starting material by one or morepurification steps (such as solvent extraction, column separation,chromatographic separation, etc.) that enrich the concentration of PALP,relative to the starting material, to an extent that PALP is thedominating component, and the remaining components do not pose anysignificant health risk and do not reduce the beneficial effects ofPALP. The term “substantially purified” should not be construed toconnote absolute purity.

The stimulatory effect of PALP on fibroblast proliferation in vitro isenhanced by pre-heating it at 65-75° C. for 30 minutes [Q. -B. She, J.J. Mukherjee, J. -S. Huang, K. S. Crilly, and Z. Kiss (2000), “Growthfactor-like effects of placental alkaline phosphatase in human fetus andmouse embryo fibroblasts,” FEBS Letters, 468, 163-167]. Although nottested yet, it is reasonable to expect that pre-heating of PALP at65-75° C. prior to its use may also enhance some aspects of itsanti-cancer effects relating to the protection of normal tissues. Thus,a step of heat-activation may be included during the final preparationof PALP for injection.

The stimulatory effect of PALP on fibroblast proliferation in vitro isalso enhanced by adding calcium and zinc to the medium [Q. -B. She, J.J. Mukheijee, J. -S. Huang, K. S. Crilly, and Z. Kiss (2000), “Growthfactor-like effects of placental alkaline phosphatase in human fetus andmouse embryo fibroblasts,” FEBS Letters, 468, 163-167]. Accordingly, thefinal preparation of PALP for injection may include 1-3 mM of acalcium-containing compound (for example, calcium chloride) and/or 1-50μM of a zinc containing compound (for example, zinc chloride or zincsulfate).

Substantially purified preparations of bone-specific, tissuenon-specific, and PALP-like (germ) alkaline phosphatase enzymes are allavailable commercially (for example, from Sigma-Aldrich). Appropriatepurification methods are known for their isolation from human blood,liver, and other organs. Also, recombinant forms of each of thesealkaline phosphatases have already been produced.

Methods of Use CC Compounds.

CCDTHT and other CC compounds may be used for the treatment of variousskin proliferative diseases, including psoriasis and skin cancer, andother proliferative diseases and cancers located in other organs. In oneembodiment suitable for the treatment of proliferative skin diseasessuch as, for example, psoriasis and skin cancer, the application istopical where the CC compound is mixed in a cream, rinse, gel, ointment,and the like. In some embodiments, the creams, ointments, and the likecontaining a CC compound can be delivered by dressings, bandages, orother similar coverings capable of releasing the therapeutic amount ofthese compounds. Such dressings can be directly placed on thehyperproliferative skin area. Oral application is another method ofdelivering a CC compound in a therapeutically effective amount. In oneembodiment of the invention, the CC compound is in the form of a tablet,gel capsule, a liquid, or the like. In each case, the CC compound ismixed with one or more carriers chosen by one having ordinary skill inthe art to best suit the goal of treatment. In yet another embodiment,the selected CC compound is mixed in a liquid biocompatible carrier,such as physiological saline (0.9% NaCl), and injected via one of thesystemic routes such as , for example, intravenous, intraarterial,intraperitoneal, subcutaneous, intramuscular, intracranial, intraportal,or intradermal. Yet another mode of application is direct injection ofCC compound-containing solution into the aberrant tissue such as a tumortissue.

The various application methods can be mixed or used alternatively. Forexample, in case of proliferative skin diseases, simultaneous topicaland systemic treatments with CCDTHT are expected to exert greatereffects than either the topical or systemic application alone. As arule, for the treatment of proliferative skin diseases, combined topicaland systemic or oral applications are recommended. For the treatment ofproliferative diseases of the internal organs such as solid and bloodcancers, either oral or systemic application of CC compounds is the mostsuitable.

The therapeutic amount of the selected CC compound that is necessary tobe delivered depends on the application method, the location of thetargeted organ, the stage of the disease, the combination of its usewith other treatments, the age of the patient, the goal of thetreatment, and other factors. The health care provider who possesses allthe required information determines the required therapeutic amount. Byan example, in case of topical application the concentration of the CCcompound in a composition will be at least about 0.01 wt.-%, and moresuitably, between about 0.1 and about 2 wt.-%. In one embodiment, thecomposition comprises about 0.2 to about 1.0 wt.-% of the activecomponent. In case of applications by injection, one dose willpreferably contain between about 10 to about 2,500 mg CC compound per m²body surface. In a suitable embodiment, the injectable compositioncontains 100 to about 600 mg CC compound per m² body surface.

The CC compound can be administered via one of the application routesfor a suitable amount of time period. The length of a suitable timeperiod, which may be 1 week, 1 month, or as many months as necessary, isindividually determined by the care provider and depends on the severityof the situation and other factors. The care provider also determinesthe frequency of the treatments. In some embodiments, the CC compoundmay be delivered three-times every 24 hours; in other cases, once perday, three-times a week, or once a week.

The CC compound-containing compositions for topical treatment mayinclude various additives or enhancers. The criterion for using anadditive/enhancer is that it increases, or at least does not decrease,the effectiveness of the active components in achieving the desiredbeneficial effect. Additives or enhancers in compositions for topicalapplications may include various ingredients, for example, preservatives(such as parabens, alcohols, phenols, essential oils, and the like),buffers, antioxidants (such as vitamin E, flavons, flavonoids,resveratrol), antimicrobials, vitamins, moisture-control agents (such asglycerine, propylene glycol, and the like), analgesics, anesthetics,anti-psoriatic agents, and anti-cancer agents. Other additives mayinclude, for example, emulsion stabilizers, preservatives, waterproofingagents, viscosity modifying agents, and the like.

For skin applications, the CC compound-containing products can alsoinclude pharmaceutically acceptable carriers or vesicles. Preferably,the carriers are non-toxic. A pharmaceutically acceptable carrier doesnot elicit an adverse physiological reaction in normal skin uponadministration and is one in which the CC compound is sufficientlysoluble to deliver therapeutically effective doses. As used herein,“pharmaceutical” is understood to encompass both human and animalpharmaceuticals. Carriers and vehicles can be included in the CCcompound products to obtain an appropriate consistency, for example,gels, lotions, cream, rinse, and the like. These products are suitableas topical compositions to control proliferation of non-healthy skincells.

Suitable carriers generally include, for example, water, acetone,ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropylmyristate, isopropyl palmitate, mineral oil, mixtures thereof, and thelike. Buffered solutions can also serve as carriers.

In some embodiments, the topical composition is a gel. The gel mayinclude as a carrier methylcellulose, sepharose, agar, vaseline orpetrolatum, agarose, gelatin, dextran, dextranpolyethylene,polyhydroxyethylmethacrylate, hydrophilic cellulose polymer,polyethylene glycol, polyvinylpyrrolidine, amylose, polyethyleneoxide,calcium alginate or combination thereof. By way of example, the selectedsterilized (filtered) CC compound can be incorporated into sterile 3% byweight methyl cellulose gel, 1% by weight agarose gel, 4% by weightgelatin gel, or 1 to 3% by weight calcium alginate. Gels of more complexcompositions can be formulated. One having ordinary skill in the artwill recognize how to vary these components to obtain sustained releaseof CC compounds.

In some embodiments, the carrier includes vaselinum flavum (yellowpetrolatum), vaselinum album (white petrolatum), or vaselinumcholesterinatum. Commercially available vaselinum cholesterinatumconsists of about 1.5 wt.-% cholesterol, about 5.0 wt.-% cerae lanae,and about 93.5 wt.-% vaselinum flavum.

The CC compound-containing compositions can be stored at roomtemperature for at least one year and at 4° C. for several years underaseptic conditions.

Embodiments of the present invention also provide CC compound-containingcompositions suitable for transdermal administration. Such compositionsare applied directly to the skin or incorporated into a protectivecarrier such as a transdermal device, i.e. a patch. Examples of suitablecreams, ointments, or the like, can be found, for example, in thePhysician's Desk Reference. Examples of suitable transdermal devices aredescribed in, for example, U.S. Pat. No. 4,818,540 to Chien et al.entitled “Transdermal Fertility Control System and Process”,incorporated herein by reference.

The CC compound-containing compositions can be made using a number ofsuitable techniques. In some embodiments, the CC compound and a carrierare mixed together within a commercial mixer to form a solution, asuspension, or the like. Various equipments are also available tomanufacture liposomal preparations (which provides for controlled,sustained release of the CC compound). In pharmaceutical compositionembodiments, methodologies for the formulation are well known, and canbe found, for example, in Remington's Pharmaceutical Sciences,Eighteenth Edition, A. R. Gennaro, Ed., Mack Publishing Co. Easton, Pa.1990, incorporated hereby by reference.

To treat hyperproliferative internal tissues, such as cancer,embodiments of the invention include CC compound in the form of a tabletor a gel-like shaped composition suitable for oral consumption orapplication through the rectum. In addition to CCDTHT, the tablet or gelmay contain any component that is presently used in the pharmaceuticalpractice to ensure firmness, stability, solubility and appropriatetaste. Any component of the tablet will be chemically inert; i.e., itwill not participate in a chemical reaction with the CC compound or theother additives. The tablet or gel may contain, in addition to theactive component and tablet-forming components, other biologicallyactive compounds such as, for example, an anticancer agent(s) or agentsthat promote(s) the actions of the CC compound. Without aiming topresent a full list, the agents increasing the effects of CC compound,for example, on cancer tissue, may include ethacrynic acid (ETA) andsimilar agents (L-buthionine-S,R-sulfoximine, diethylmaleate,2-cyclohexene-1-one, 1-chloro-2,4-dinitrobenzene) which decrease thecellular level of glutathione, zinc, or cadmium at concentrations whichdecrease the proliferation of non-healthy cells but are non-toxic tonormal cells, pyrrolidinedithiocarbamate and any other chelator whichcan bind and carry vitally important metal ions such as, for example,zinc, copper, or iron, any agent that can reverse multidrug or otherforms of drug resistance, or any other anti-proliferation/anticanceragent or drug which acts additively or synergistically with the CCcompound and is suitable to mix with a CC compound to make a tablet, agel capsule and the like for oral delivery.

Appropriate solutions of the selected CC compound, which may includeother anti-cancer (anti-proliferation) drugs or additives, can also beapplied via other routes including intravenous, intramuscular,intraperitoneal, intradermal, intraarterial, intracranial, intraportal,and subcutaneous injections or aerosol. Yet another mode of applicationis direct injection into the aberrant tissue such as a tumor tissue. Forall injection methods, the CC compound can be dissolved in anyappropriately accepted solvent or medium which provides good solubilityand which is physiologically compatible. One generally accepted mediumis physiological saline (0.9% NaCl). The compound, CCDTHT, and most ofthe other CC compounds including CCcompound26, are sufficiently solublein water for injection.

For both topical applications on the skin and injection treatments ofinternal non-healthy tissues, the CC compound-containing compositionsmay be used as the sole method of medication, or they may be used incombination with other treatments. For example, it is envisioned that inthe case of psoriasis, the CC compound-containing composition may beused either alone or in combination with available anti-psoriasis agentsacting, in most cases, by suppressing the actions of specific immunecells. The same may be true for some cancers, such as ovarian cancer,where the selected CC compound may be sufficiently effective alone(based on in vitro data); however, it could become even more effectivewhen cisplatin or other anti-cancer agents are used simultaneously.Similarly, in the elderly, who may not tolerate highly toxicchemotherapy, treatments with the CC compound alone may be morebeneficial than strongly cytotoxic drugs to prolong life without causingmajor side effects. However, in most cases it is recommended that theselected CC compound be used together with an anti-proliferation agent,such as anti-cancer agent, to enhance the efficacy and decrease thetoxicity of the latter agents.

In case of certain cancers CC compound-containing compositions may beused between two series of chemotherapy (which are usually 2-3 weeksapart), to allow patients to recover from the side effects ofchemotherapy and to ensure at least partial suppression of tumor growthbetween the chemotherapeutic treatments. However, the CCcompound-containing compositions may also be used simultaneously withother chemotherapeutic agents, allowing the latter agents to be used atlower concentrations with less toxic side effects. The sequence oftreatments with chemotherapeutic agents and CC compound-based formulas,the length of each treatment, and the dosing of CC compound and variousanticancer agents will be determined, based on previous experience,individually by the health care provider.

CC compound-containing compositions may be used in combination oralternatively with anti-proliferation treatments other thanchemotherapy, such as radiation and surgery, for example. In oneregimen, the CC compound-containing cream may be used after radiationand/or surgical procedures as an after treatment to prevent localrecurrence of skin cancer. In another regimen, CC compound-containingsolution is applied by one of the injection methods after surgicalremoval of tumor from an internal organ or after completing a course ofchemotherapy or radiotherapy to prevent recurrence of the primary tumoror development of secondary tumors.

It is now generally accepted that most, if not all, tumors areheterogeneous due to their multiclonal origin. This means that thenature and level of aberrations are somewhat or significantly differentin different sub-populations of the tumor-forming cells. In practicalterms, this means that subsets of cells in the same tumor may notrespond similarly to the same treatment. In the present invention,different CC compounds are shown to exert in some cases differenteffects against different cancer cell types. For example, different CCcompounds inhibit the proliferation of the MEL-24 and MEL-28 humanmelanoma cells. Therefore, in one embodiment of treating human ormammalian tumors two or more CC compounds may be used in thecompositions used for oral, local, or systemic applications. Suchcompositions then can be used in combination with otheranti-proliferation treatments such as chemotherapy, radiation, surgery,or suppression of the immune system.

Alkaline Phosphatase.

The model alkaline phosphatase used as an anticancer agent inembodiments of the invention is the placental form of alkalinephosphatase (PALP) produced by the placenta during pregnancy. However,all alkaline phosphatases commonly express an alkaline phosphataseenzyme activity that may play a role in the PALP's anticancer effects.Therefore, other alkaline phosphatase enzymes may mimic, at least inpart, the anticancer effects of PALP.

The anticancer effect of PALP has three components; i.e., it (a) alonedecreases the size of certain tumors and also enhances the similareffects of anti-cancer agents, including CC compounds, (b) prevents,partially or fully, the decrease in body weight induced by chemotherapy,and (c) increases survival time in certain tumor models. For thetreatment of most cancers with an alkaline phosphatase one of thesystemic application routes is recommended.

Therapeutically effective amounts of sufficiently highly purified orhomogeneous PALP or another alkaline phosphatase may be used as theactive component in the compositions described herein. Alternatively,preparations containing synthetic protein or its active derivative, or arecombinant form of alkaline phosphatase or its active derivative, maybe employed as the active component. The term “active” means that thepreparation of intact alkaline phosphatase, or a fragment of it, exertsat least one of the above listed three anticancer effects. The term“therapeutically effective amount” in this specification indicates adosage that is effective in exerting at least one of the above threeanticancer effects.

A composition comprising the active protein component may beadministered by one of the injection methods including intradermal,subcutaneous, intravenous, intraperitoneal, intraarterial, intracranial,and intramuscular applications. The injectable form of the compositionis comprised of a therapeutically effective amount of PALP or anotheralkaline phosphatase or an active derivative thereof.

For injection of a composition comprising the active alkalinephosphatase component, the carrier can be any physiologically acceptablecarrier that does not cause an undesirable physiological effect and iscapable of ensuring proper distribution of the active components in thetreated tissue. The active components are dissolved or dispersed in thephysiologically acceptable carrier. Examples of carriers includephysiological saline and phosphate-buffered saline. Alternatively, theactive alkaline phosphatase component may be enclosed in liposomes suchas immunoliposomes, or other delivery systems or formulations that areknown in the art may be employed. By way of example, the active proteincomponent can be readily dissolved in physiological saline (0.9% NaCl),or in any other physiologically competent carrier, to yield a solutionfor injection.

An injectable composition may be prepared by dissolving or dispersing asuitable preparation of the active protein component in the carrierusing conventional methods. As examples only, one embodiment of theinvention includes PALP in a 0.9% physiological salt solution to yield atotal protein concentration of 10 mg/ml. Another embodiment includesPALP in a 0.9% physiological salt solution to yield a total proteinconcentration of 200 mg/ml.

The injectable composition may be modified by any number of additivesand enhancers, as listed above for the topical application, that may bedissolved or suspended in the composition and that are expected topromote the anticancer effects of the alkaline phosphatase component ordiminish any potential side effect.

In one embodiment of the method, the mode of injection is selected fromintradermal, intravenous, subcutaneous, intramuscular, intraarterial,intracranial, intraportal and intraperitoneal. The mode of injection isselected to provide either local delivery to some tumors such as livertumor (intraarterial), or systemic delivery to other cancers via theblood supply (such as intravenous, intraperitoneal and subcutaneousapplications).

A common way to express a suitable dosage for systemic administration isgrams of the active agent(s) per square meter of body surface area forthe subject. Those having ordinary skill in the art are familiar withthe formulas used for estimating a human subject's body surface area,based on the human's height (in cm) and mass (in kg).

In case of intravenous, intraarterial, intramuscular, intraperitoneal,intraportal, intracranial or subcutaneous application, the subject maybe administered a total of about 0.02 to 2.5-g PALP per m² body surfaceonce daily. In another embodiment, a subject may be administered byintravenous, intraarterial, intramuscular, intracranial,intraperitoneal, intraportal, or subcutaneous application a total ofabout 0.02 to 2.5-g PALP per m² body surface twice or three timesweekly. Alternatively, the subject may be administered a total of about0.02 to 2.5-g PALP per m² body surface once a week or biweekly byintravenous, intraarterial, intramuscular, intracranial,intraperitoneal, intraportal, or subcutaneous application. Since thehalf-life time of PALP is relatively long (5-6 days), in one embodimentof the invention, the protein is applied twice a week or once a week.Again, concerning the effective tolerable dose, an importantconsideration is whether alkaline phosphatase is used alone or used aspart of a more complex regimen involving other anticancer agents aswell. For example, if the subject is simultaneously or alternativelytreated with both alkaline phosphatase and another therapy, theeffective tolerated amount of the former may be less compared to aregimen when the subject is treated with alkaline phosphatase alone.

Use of CC Compound and PALP in Combination.

In various tumor models, the CC compound, CCDTHT (CCcompound3), and PALPcommonly reduced tumor size, enhanced the effects of chemotherapeuticagents on tumor size, decreased or fully prevented weight loss inducedby chemotherapeutic agents, and increased survival. In some cases, asdemonstrated under “Examples”, the combined effects of CCDTHT and PALPwere greater than their individual effects. These findings indicate thatthere are cases when using a selected CC compound and alkalinephosphatase in combination for the treatment of animal or human tumorswill exert greater anti-tumor effects than using them alone. The same islikely to be true for other proliferative diseases as well.

The combined treatment with CC compound and alkaline phosphatase mayoccur in the absence or presence of other treatments performedsimultaneously or alternatively. The combined treatment with CC compoundand alkaline phosphatase may also occur prior to or after the othertherapies. For example, prior treatment with relatively non-toxic CCcompound and PALP could serve to shrink the tumor to a more manageablesize for the subsequent surgery and/or radiation. The combined treatmentwith CC compound and PALP may also be performed as an after treatmentfollowing surgical removal and/or radiation treatment of a tumor toreduce local or remote (metastasis) recurrence.

Since CCDTHT and PALP act via independent mechanisms and therefore donot interfere with each other's actions, both agents may be used at thesame therapeutically effective doses as recommended when usedindividually. The same is true for the regimens; similar regimensdeveloped for the individual use of CC compound and alkaline phosphatasemay be employed when they are used in combination.

For applications via injection, the injectable preparation mayoccasionally contain both the CC compound and alkaline phosphatase.However, in this latter case separate application of the CC compound andalkaline phosphatase is more likely because the former requires morefrequent applications than the latter. The CC compound-containinginjectable preparation may also contain other anticancer agent(s), orthe latter may be administered separately.

In addition to injection methods, CC compounds may also be administeredorally or topically, independent of the injected alkaline phosphatase.

For the treatment of most cancers, a preferred regimen includesperiodical injection or oral administration of an establishedchemotherapeutic agent accompanied by daily oral administration of CCcompound and once or twice a week injection of PALP. Again, thistreatment may be combined with surgery and/or radiation, or by any othertherapy, either prior to or after the primary therapy.

EXAMPLE 1 Synthesis ofN,N-diethyl-N-methyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]-ethanaminiumiodide (CCcompound3 or CCDTHT)

This product has been chemically synthesized via the followingintermediates:

2-[(4-methylphenyl)thio]-benzoicacid→2-methyl-9H-thioxanthen-9-one→2-(bromomethyl)-9H-thioxanthen-9-one)→2-{[2-diethylamino)ethoxy]methyl}-9H-thioxanthen-9-one→N,N-diethyl-N-methyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]-ethanaminiumiodide.

Synthesis of 2-[(4-methylphenyl)thio]-benzoic acid (intermediate 1):2′-iodo benzoic acid (19.84 g) and p-thiocresol (9.92 g) were dissolvedin 120 ml of dimethylformamide; this was followed by the addition of 0.5g of copper powder, 0.5 g of Cleland's reagent (threo-1-1,4dimercapto-2,3-butanediol) and 29.52 g of hot K₂CO₃. The mixture washeated, refluxed (in an oil bath) with continuous vigorous mixing, andcooled to room temperature. Then the mixture was filtered, and the solidfraction was washed three-times with dimethylformamide. The solidfraction was removed, and the combined filtrate was evaporated (using aRotadest equipment). The resulting solid fraction was dissolved inwater, filtered, placed on ice, and pH adjusted to 1.0 with H₂SO₄. Theresulting white precipitate was filtered and washed with water until thepH reached 7.0. The efficacy of this reaction is about 80%. The meltingpoint of intermediate 1 is 218-219° C.

Synthesis of 2-methyl-9H-thioxanthen-9-one (intermediate 2):Intermediate 1 (17.0 g) and polyphosphoric acid (110 g) were mixed andthen heated to ˜150° C. with vigorous mixing for 5 hours. After coolingthe mixture to room temperature, ice-cold water was added to it. Themixture was extracted three times with 150 ml of ethylacetate. Thecombined organic phases were first washed three times with 150 ml of 5%Na₂CO₃, then once with water (150 ml), and once with saturated NaCl (150ml), followed by drying the washed organic phase over water-free solidNa₂SO₄. After removing the solvent, the yellow material wasre-crystallized from ethanol. The efficacy of this reaction is 65%. Themelting point of intermediate 2 is ˜123.5° C.

2-(bromomethyl)-9H-thioxanthen-9-one) (intermediate 3): Intermediate 2(20.8 g), 1,3′-dibromo-5,5′-dimethylhydantoin (13.5 g) andbenzoylperoxide (4.4 g) were suspended in 800 ml of absolute CCl₄(carbon tetrachloride), and then the suspension was refluxed for 20hours with continuous and rigorous mixing. The mixture was cooled to˜10° C. and filtered. The remaining solid material was washed threetimes with 100 ml water (˜75° C.) and then re-crystallized from hotacetone. The solid material was washed with cold acetone. The efficacyof this reaction is 51%. The melting point of intermediate 3 is 196-197°C.

2-{[2-diethylamino)ethoxy]methyl}-9H-thioxanthen-9-one (intermediate 4):2′-diethylaminoethanol (1.17 g) was dissolved in 50 ml of absolutexylol; then during constant vigorous mixing and warming to 35-40° C.0.24 g of solid sodium was added in small portions followed by mixingand warming of the mixture for one hour. Intermediate 3 (1.5 g) wasadded to the mixture in small portions, and the whole mixture was mildlyrefluxed for 56 hours. The solid material was removed by filtering themixture. The organic phase was washed four times with 150 ml water.Then, the water was removed by filtration through solid water-freeNa₂SO₄. After distillation (to remove xylol), the resulting oily,yellow-brown material was not yet pure as determined by thin layerchromatography (Kiesel gel) using benzol:ethanol:water (50:15:1.5, v/v)as solvent. The mixture was fully purified through the following steps.The mixture was first dissolved in an (CH₃)₂CO (acetone)/absoluteethanol (1:1, by volume) mixture followed by the addition ofconcentrated HCl until the solid material (HCl salt) was formed. Thesolid precipitate was washed with cold acetone. The HCl salt ofintermediate 4 was dissolved in ethanol:water (10:90, v/v), and thesolution was made basic (pH 14) by the addition of solid Na₂CO₃. Theoily material was extracted with ethylacetate and then dried over solidNa₂SO₄. Intermediate 4 is 99-100% pure as determined by ¹H-NMR and thinlayer chromatography. The yield of intermediate 4 is ˜55%.

Synthesis ofN,N-diethyl-N-methyl-2-[(9-oxo-9H-thioxanthen-2-yl)methoxy]-ethanaminiumiodide (CCcompound3 or CCDTHT): Intermediate 4 (1.0 g) was dissolved in5 ml of absolute (CH₃)₂CO (acetone) followed by the addition (at roomtemperature) of methyl iodide (1 ml). Within 1-2 minutes, a white solidmaterial (CCDTHT) was formed. After keeping the mixture at 4° C.overnight, the solid material was filtered and was washed twice with 100ml of (CH₃)₂CO. CCDTHT is a light-yellow crystallized material. Nocontaminant can be seen by NMR or thin layer chromatography (TLC;benzol:ethanol:water=50:15:1.5, by volume as solvent) which wasreasonable to expect because intermediate 4 was already ˜99-100% pure.Also, the precursor and the final product (CCDTHT; Rf=0.35) arephysically well separated by TLC, and in the last reaction only oneproduct, i.e. CCDTHT, can be formed. The melting point of CCDTHT is˜225° C.

EXAMPLE 2 Synthesis of CCcompound17,N,N,N-trimethyl-3-(9-hydroxy-9H-thioxanthen-9-yl)-propane-1-aminiumiodide

Thioxanthone (2.76 g, 13 mmol) was dissolved in 40 ml tetrahydrofuran.The thioxanthone solution was added drop-wise, during intensive mixing,to a solution containing 27 mmol of Grignard reagent (made by reactingmagnesium first with dibromidemethane and then with diethylaminopropilchloride by a method well known in the art), 50 ml of diethylether and50 ml of tetrahydrofuran. The mixture was refluxed for 2.5 hoursfollowed by constant mixing at room temperature for 16 hours. Then, themixture was added to 200 ml of ice-cold saturated ammonium chloride andthe solution was extracted 3-times with 100 ml of ethylacetate. Afterdrying the organic phase over water-free sodium sulfate, the solvent wasremoved by distillation. The resulting yellow product (C₁₈H₂₁NOS) wasfirst re-crystallized from an ethanol/acetone mixture, then re-dissolved(0.6-g) in I0 ml of absolute acetone+2 ml methyl iodide. The mixture wasfirst kept for 24 hours at room temperature and then for 2 days at 4° C.in a closed glass tube (no contact with air). Then, the precipitatedcrystals were filtered and washed with absolute acetone. The finalproduct (C₁₉H₂₄JNOS; Mw+ 441.385) was dried under vacuum in the presenceof P₂O₅.

EXAMPLE 3 Synthesis of CCcompound18,3-(9-hydroxy-9H-selenoxanthen-9-yl)-N,N,N-trimethylpropane-1-aminiumiodide

In the first step, selenoxanthen-9-one was made by well knownconventional steps involving the synthesis of 2-carbonyl selenodiphenolby reacting selenophenol with iodobenzoic acid followed by ring closurein the presence of concentrated H₂SO₄. The synthetic steps were thenperformed as described for the synthesis of CCcompound17 in Example 2,except that thioxanthone was replaced with selenoxanthen-8-one.

EXAMPLE 4 Synthesis of CCcompound19,N,N,N-trimethyl-3-(9H-thioxanthen-9-ylidene)-propane-1-aminium iodide

CCcompound17 was dissolved in a mixture of 35 ml distilled water and 3ml of concentrated H₂SO₄ followed by reflux of the solution for 24hours. Then, the solution was diluted with 20% (w/v) potassium hydroxideuntil the pH reached a value between 11 to 12. The resultant whiteemulsion was extracted 3-times with 100 ml of ethyl acetate. This wasfollowed by washing the organic phase with water until the pH becomeneutral and drying the organic phase over water-free sodium sulfate.After distillation under vacuum, the product was a light brown oilymaterial that was further enriched in the form of HCL salt. The oilymaterial was dissolved in 20 ml of absolute acetone to which a mixtureof ethanol and HCl (ethanol saturated with HCl) was added until crystalformation was induced. Then, the precipitated crystals (C₁₈H₂₀ClNS) werefiltered, washed with acetone, and dried at room temperature. Thecrystals were dissolved in water followed by the addition of 50%potassium hydroxide until pH 11 was reached. The precipitate (C₁₈H₁₉NS)was filtered, washed with acetone and dried. This basic compound(C₁₈H₁₉NS) was dissolved in 10 ml of absolute acetone followed by theaddition of 1.2 ml of methyl iodide to the mixture. The crystals thatare almost immediately formed at room temperature were kept at 4° C. for24 hours and then filtered. The precipitate was washed with acetone anddried at room temperature. The molecular weight of the final product(C₁₉H₂₂JNS) was 423.

EXAMPLE 5 The synthesis of CCcompound20,N,N,N-trimethyl-3-(9H-selenoxanthen-9-ylidene)-propane-1-aminium iodide

The synthesis of this compound was performed similar to that ofCCcompound19 as described in Example 4, except that in this caseCCcompound18 was used as the starting material.

EXAMPLE 6 The synthesis of CCcompound23,N,N-diethyl-N-methyl-3-(2-methyl-3-9H-thioxan then-9-ylidene)-propane-1-aminium-iodide

The synthesis involved the following steps:

2-methylthioxanthon→9-[3-(diethylamino)propyl]-2-methyl-9H-thioxanthen-9-ol→N,N-diethyl-N-[3-(2-methyl-9H-thioxanthen-9-ylidene]-propylamine→N,N,-diethyl-N-methyl-3-(2-methyl-9H-thoxanthen-9-ylidene)-propane-1-aminium-iodide.

Synthesis of 9-[3-(diethylamino)propyl]-2-methyl-9H-thioxanthen-9-ol(intermediate 1): 2-methylthioxanthone (4 g; 17.6 mmol) was dissolved in50 ml of tetrahydrofuran. The thioxanthone solution was added drop-wise(over a 30 min period), during intensive mixing, to a solution (125 ml)containing 60 mmol of Grignard reagent (made as described under Example2) and 100 ml of tetrahydrofuran. The mixture was refluxed for 5 hoursfollowed by constant mixing at room temperature for 16 hours. Then themixture was added to 500 ml of ice cold 50% saturated ammonium chloridefollowed by the collection of the organic phase. The water phase wasextracted 3-times with 100 ml of ethylacetate. After drying the combinedorganic phase over water-free sodium sulfate, the solvent was removed bydistillation. The resulting light yellow product (91% yield) was 99-100%pure as determined by NMR and thin layer chromatography(benzol:ethanol:water=50:15:3, by volume as solvent; Rf=0.41).

Synthesis of N,N-diethyl-N-[3-(2-methyl-9H-thioxanthen-9-ylidene]-propylamine (intermediate 2): Intermediate 1 (4 g) was dissolved in a mixtureof 94 ml water+5.37 ml concentrated H₂SO₄; then the mixture was refluxedfor 1.5 hour with constant mixing. The mixture was added to 100 ml ofice-cold NaOH (pH 12) followed by extraction with 3×70 ml ethylacetate.Then 100 ml of 1 M HCl was added to the combined organic fraction tofacilitate the transfer of the product from the organic phase to theacidic water phase. Next, solid K₂CO₃ was added to the acidic waterphase to adjust the pH to about 12; this step was followed by extractionof the mixture with ethylacetate (3×70 ml). The combined organic phasewas dried over water-free Na₂SO₄, followed by removal of the solvent bydistillation. The resulting solid product was first dissolved inacetone/absolute ethanol, 1:1 by volume, mixture followed by theaddition of concentrated HCl until the solid material (HCl salt) wasformed. The solid precipitate was washed with cold acetone.

Synthesis ofN,N-diethyl-N-methyl-3-(2-methyl-3-9H-thioxanthen-9-ylidene)-propane-1-aminium-iodide:

Two grams of intermediate 2 was dissolved in 10 ml water, followed byadjusting the pH to about 12 with 2 N NaOH; this mixture was thenextracted with 2×20 ml of ethylacetate. The combined organic phase wasdried over water-free Na₂SO₄, followed by removal of the solvent bydistillation. The resulting material was dissolved in 10 ml of absoluteacetone followed by the addition of 2 ml of methyl iodide. The mixturewas first left for 12 hours at room temperature and then for 12 hours at4° C. in a closed glass tube (to avoid contact with air). The solidproduct was filtered and washed with absolute acetone. The final productis a light yellow crystal which was 99-100% pure as verified by thinlayer chromatography (TLC; benzol:ethanol:water=50:15:1.5, by volume assolvent) and NMR spectra.

EXAMPLE 7 Synthesis ofN,N,N-triethyl-3-(2-methyl-9H-thioxanthen-9-ylidene)-propane-1-aminium-iodide(CCcompound25)

CCcompound25 was synthesized by the same procedure described forCCcompound23, except that in the last step 2 ml of ethyl iodide, insteadof methyl iodide, was used.

EXAMPLE 8 Synthesis ofN,N-diethyl-N-allyl-3-(2-methyl-9H-thioxanthen-9-ylidene)-propane-1-aminium-bromide(CCcompound26)

CCcompound26 was synthesized by the same procedure described forCCcompound23, except that in the last step 2 ml of allyl bromide,instead of methyl iodide, was used.

EXAMPLE 9 Purification and Spectrophotometric Assay of PALP

Human PALP (Type XXIV, 1020 units of total activity) in a partiallypurified form was obtained commercially from Sigma-Aldrich. A butanolextraction of placental tissue, followed by ammonium sulfateprecipitation and two chromatographic steps, was performed bySigma-Aldrich to obtain the partially purified material. Butanolextraction inactivates most, if not all, of the other placentalproteins, including growth factors, but does not reduce the mitogenic orenzymatic activity of PALP.

As determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), the partially purified PALP obtained fromSigma-Aldrich (denoted “commercial PALP” herein) was not homogeneous andcontained other proteins. FIG. 1 shows a picture of a gel separation ofa preparation comprising commercial PALP without further purification,and other preparations of PALP of increasing purity. Separation ofproteins was performed by conventional SDS-PAGE, and proteins werestained with coomassie blue stain. Lane 1 contains various molecularmass standards for comparison. Lane 2 represents a preparationcontaining commercial PALP with a strong 52 kDa band representingα₁-antitrypsin and another strong 66 kDa band representing a mixture ofPALP and albumin. Lanes 3 and 4 represent preparations comprisingcommercial PALP material after further purification steps (describedbelow), and lane 5 represents a preparation of homogeneous PALP obtainedby the complete purification procedure described below.

A purification procedure consisting of several steps was performed tofurther purify the commercially obtained PALP to homogeneity. A similarpurification procedure described by Mukherjee et al. [She, Q. -B.,Mukheijee, J. J., Huang, J. -S., Crilly, K. S. and Kiss, Z. (2000),“Growth factor-like effects of placental alkaline phosphatase in humanand mouse embryo fibroblasts,” FEBS Lett. 469, 163-167] was used, exceptthat in embodiments of the invention the last chromatographic step wasperformed twice.

A solution of commercial PALP was prepared by dissolving 350 mg ofcommercial PALP into 10 ml of buffer A (0.1 M sodium acetate, 0.5 MNaCl, 1 mM MgCl₂, 1 mM CaCl₂, adjusted to pH 6.5). This solution wasthen further purified by successive Concanavalin A-Sepharose andQ-Sepharose chromatography, essentially following the proceduredescribed elsewhere [Chang, T. -C., Huang, S. -M., Huang, T. -M. andChang, G. -G. (1992), “Human placenta alkaline phosphatase: An improvedpurification procedure and kinetic studies,” Eur. J. Biochem. 209,241-247].

The solution was run through a Concanavalin A-Sepharose column followedby an elution step using buffer A as solvent. For elution, buffer Aincluded 50 mM a-methyl-D-mannopyranoside. The active fractionscollected from the effluent were pooled and dialyzed against buffer B(50 mM Tris-HCL at pH 7.7). SDS-PAGE separation of the collected anddialyzed fraction is shown in lane 3 of FIG. 1.

The collected and dialyzed fraction from the previous step was thenpassed through a Q-Sepharose column. The fraction of interest was elutedwith buffer B using a linear gradient of 0-250 mM potassium phosphate ata pH of 7.5. The active fractions from the Q-Sepharose column werepooled and dialyzed against phosphate-buffered saline and concentratedby Amicon ultrafiltration. SDS-PAGE separation of the collected anddialyzed fraction is shown in FIG. 1 in lane 4, which demonstrates thatat least two major proteins are still present in the fraction afterdialysis.

Then, the collected and dialyzed fraction from the previous step waspurified to homogeneity by t-butyl hydrophobic interactionchromatography (HIC). Prior to adding the fraction to the t-butyl HICcolumn, the fraction was made 2 M in ammonium sulfate, and pH wasadjusted to 6.8. The 5 ml bed volume t-butyl HIC cartridge (BIO-RAD,Hercules, Calif.) was connected to a fast performance liquidchromatography (FPLC) system from PHARMACIA (Peapack, N.J.). Thefraction was introduced to the HIC column, and the column was elutedwith buffer C (100 mM sodium phosphate buffer, 2 M ammonium sulfate atpH 6.8). The column was eluted with buffer C until a firstprotein-containing fraction completely eluted, and then a negativegradient of 2 M-to-0 M ammonium sulfate in 100 mM sodium phosphate at pH6.8 was passed over the column. The negative linear gradient was used toelute a second protein-containing fraction, which contained theenzymatically active PALP protein.

The enzymatically active fraction from the HIC separation was dialyzedagainst phosphate buffered saline and concentrated by Amiconultrafiltration. Presence and purity of the PALP enzyme in the fractionwas confirmed by SDS-PAGE. After electrophoretic separation, the gel wasstained using coomassie blue or silver stain for visual observation ofprotein bands. In about 50% of cases a single protein band was observedwith an approximate molecular weight of 66 kDa. In the other 50% ofcases the PALP fraction still was slightly contaminated byα1-anti-trypsin, probably reflecting the higher amount of this proteinin the starting commercial preparation. In these latter cases, the lastchromatography step was repeated to entirely remove α1-anti-trypsin. Thepure PALP was further identified by sequence analysis performed by theMayo Clinic Protein Core Facility (Rochester, Minn., USA).

PALP enzyme activity was assayed using a spectroscopic method bymonitoring the hydrolysis of 4-nitrophenylphosphate (as an increase inabsorbance at 410 nm) at room temperature (22° C.) as describedelsewhere [Chang, G. -G., Shiao, M. -S., Lee, K. -R. and Wu, J. -J.(1990), “Modification of human placental alkaline phosphatase byperiodate-oxidized 1,N⁶-ethenoadenosine monophosphate,” Biochem. J. 272,683-690]. Activity analysis of 5-10 μg purified enzyme was performed in1 mL incubation volume containing 50 mM Na₂CO₃/NaHCO₃, 10 mM MgCl₂, 10mM 4-nitrophenylphosphate at pH 9.8. The extinction coefficient of4-nitrophenol was taken as 1.62×10⁴ M⁻¹ cm⁻¹. An enzyme activity of 1 U(unit) is defined as 1 μmol substrate hydrolyzed/min at 22° C. at pH9.8.

EXAMPLE 10 Determination of Cell Viability

Cells that lost viability do not synthesize DNA. To quantify cell death,around the time when significant and characteristic morphologicalchanges took place, cells were pulse-labeled for 10 minutes with[³H]thymidine to measure DNA activity as described elsewhere [Tomono, M.and Kiss, Z. (1995), “Ethanol enhances the stimulatory effects ofinsulin and insulin-like growth factor-I on DNA synthesis in NIH 3T3fibroblasts,” Biochem. Biophys. Res. Commun. 208, 63-67]. Also, cellswere collected as suspensions using trypsin, an established method incell biology, followed by re-plating of cells in fresh tissue culturemedium. If no viable cells were obtained after one week in culture, itmeant that the cells at the time of re-plating were dead. Survivalvalues obtained with untreated cells were considered 100%. For thedetermination of relative number of viable cells after treatments, inmost cases the MTT assay was used. This calorimetric assay is based onthe ability of living cells, but not dead cells, to reduce 3-(4,5-dimethyl thiazol-2-yl)-2, 5-diphenyltetrazolium bromide [Carmichael,J, De Graff, W. G., Gazdar, A. F., Minna, J. D. and Mitchell, J. B.(1987), “Evaluation of tetrazolium-based semi-automated calorimetricassay: Assessment of chemosensitivity testing,” Cancer Res. 47,936-942]. For this assay, cells were plated in 96-well plates, and theMTT assay was performed both in untreated and treated cell cultures. TheMTT assay also was performed at the start of treatments to allowassessment of proliferation rates in the control and treated cellcultures.

EXAMPLE 11 Cell Lines and Cell Culture Reagents

Fetal bovine serum (FBS) and all tissue culture media were bought fromLife Technologies (formerly GIBCO BRL) (Rockville, Md.). Except HaCaTkeratinocytes (which were provided by the Institute of Dermatology,Szeged University, Szeged, Hungary) as well as MCF-7 and MCF-7/MDR1cells (provided by the National Institute of Health, Bethesda, Md.), allother cell lines were provided by the American Type Culture Collection[Rockville, Md].

EXAMPLE 12 Determination of Effects of CCDTHT on Choline Metabolism inCancer Cells

MEL-28 and MCF-7 cells were cultured in MEM medium supplemented with 10%fetal bovine serum (FBS) and in DMEM medium supplemented with 10% FBS,respectively. The AN3CA human endometrial adenocarcinoma cells weremaintained in Eagle's MEM containing 10% FBS. Cancer cells were grown upto confluency in 12-well plates in 10% fetal-bovine serum-containingmedium, then incubated in the presence of 1 μCi of [methyl-¹⁴C]choline(bought from Amersham; Piscataway, N.J.) for 2 hours. Cells were firstrapidly (within 30 seconds) washed three times with 4 ml of serum-freemedium, then extracted with ice-cold methanol. This was followed by thedetermination of cellular free radiolabeled choline and phosphocholine(PCho) by column chromatography as described earlier [Kiss, Z. andCrilly, K. S. (1995), “Tamoxifen inhibits uptake and metabolism ofethanolamine and choline in multidrug-resistant, but not indrug-sensitive, MCF-7 human breast carcinoma cells,” FEBS Lett. 360,165-168], and the separation and determination of radiolabeledphosphatidylcholine (PtdCho) by thin layer chromatography as indicatedin Kiss [Kiss, Z. (1996), “Direct proof that phorbol ester acceleratesthe use of choline phosphate for phosphatidylcholine synthesis in intactcells,” Arch. Biochem. Biophys. 335, 191-196].

EXAMPLE 13 Development and Treatment of Tumor Models

Tumors were developed in first generation hybrid BDF1 (C57 B1female×DBA/2 male) adult female mice kept at specified pathogen free(SPF) hygienic level. Suspensions of HL-60 human leukemia cells, orapproximately 0.1 cm³ volume of tumor tissue derived from HT-168 humanmelanoma, PC-3 human prostate adenocarcinoma, T47D human breastcarcinoma, MXT mouse mammary cancer, or mouse B16 melanoma cells wereimplanted subcutaneously to develop the tumors. After 12-18 days whenthe tumor-bearing mice were first treated, the size of the tumors was inthe 0.2-0.4 cm³ range. All compounds used were dissolved inphysiological (0.9%) saline (NaCl), and the agents, alone or incombinations, were applied subcutaneously or intraperitoneally in 50 μlvolume. Tumor volume was determined by calipers in three dimensions;this technique is well known to one having ordinary skill in the art.Tumor volume was calculated according to the generally accepted formula:V=a²×b×π/6, where “a” and “b” mean the shortest and longest diameter,respectively, of the measured tumor.

Examples showing that CCDTHT induces the death of cancer cells asindicated by their rounded-up morphology.

EXAMPLE 14 CCDTHT Induces the Death of Human Melanoma MEL-28 Cells

About 60% confluent cultures of MEL-28 cells, cultured in 12-well platesin MEM medium supplemented with 10% FBS, were either untreated (FIG.2A), or were treated with 100 μM CCDTHT (CCcompound3) for 72 hours (FIG.2B). The pictures, taken 72 hours after the treatment, show that whilethe control cells reached the confluent state and remained healthy (FIG.2A), the cell culture treated with CCDTHT (FIG. 2B) remained at about60% confluent with practically all cells rounded up, indicating celldeath via the apoptotic pathway. This experiment was repeated four timeswith similar results. In parallel experiments, cells treated for 72hours with CCDTHT did not synthesize detectable amount of DNA asconcluded from the absence of incorporation of radiolabeled thymidineinto DNA. This indicates that the observed morphological changes wereindeed associated with cell death.

EXAMPLE 15 CCDTHT Induces the Death of Estrogen Positive Human BreastCancer MCF-7 Cells

About 50% confluent cultures of MCF-7 cells, cultured in 12-well platesin Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS,were either untreated (FIG. 3A), or were treated with 100 μM CCDTHT(CCcompound3) for 72 hours (FIG. 3B). The pictures, taken 72 hours afterthe treatment, show that the control cells reached a healthy confluentstate (FIG. 3A). In contrast, the MCF-7 cell culture treated with CCDTHT(FIG. 3B) remained sub-confluent with practically all cells rounded up,again indicating cell death via the apoptotic pathway. This experimentwas repeated three times with similar results. In some experiments,cells from the CCDTHT-treated cultures were seeded into 96-well plates,and the MTT assay was performed 96 hours later to test for viable cells.The MTT assay did not detect viable cells, indicating that (i) the abovetreatment with CCDTHT indeed killed practically all MCF-7 cells, andthat (ii) the morphological pictures faithfully indicated cell death inCCDTHT-treated cell cultures.

EXAMPLE 16 CCDTHT Induces the Death of Estrogen-Positive Human BreastCancer T47D Cells

About 40% confluent cultures of T47D cells, cultured in 12-well platesin RPMI 1640 medium supplemented with 0.2 IU bovine insulin and 10% FBS,were either untreated (FIG. 4A), or were treated with 100 μM CCDTHT(CCcompound3) for 72 hours (FIG. 4B). The pictures, taken 72 hours afterthe treatment, show that the control cells reached a healthy confluentstate (FIG. 4A), while the T47D culture treated with CCDTHT (FIG. 4B)remained about 40% confluent with practically all cells rounded up,indicating cell death via the apoptotic pathway. This experiment wasrepeated three times with similar results. In some experiments, cellsfrom the CCDTHT-treated cultures were seeded into 12-well plates todetermine DNA synthesis 96 hours later; no DNA synthesis could bedetected at that time point indicating that the above treatment withCCDTHT indeed killed practically all T47D cells.

Examples demonstrating the effects of CC compounds on the viability ofnormal and cancer cells using the MTT assay.

EXAMPLE 17 Comparison of Effects of CCcompound1 and CCcompound3 (CCDTHT)on the Viability of Normal and Cancer Cells

The NIH 3T3 (normal) mouse fibroblast, A431 human epidermoidadenocarcinoma, and MCF-7 human breast cancer cells were cultured inDMEM medium containing 10% FBS. The MEL-28 human melanoma cells werecultured in MEM medium containing 10% FBS. The HT-29 human colonadenocarcinoma cells were cultured in McCoy's 5a medium containing 10%FBS. The A549 human lung carcinoma cells were maintained in Ham's F12Kmedium containing 10% FBS. Cells were seeded into 96-well plates andtreated at ˜70-90% confluency. Two hours before treatments the mediumwas changed for fresh 10% serum medium, and then the cells wereincubated for 72 hours in the presence of 0-200 μM of CCcompound1 () orCCcompound3 (CCDTHT) (▴), as indicated. The MTT assay was used todetermine cell viability after treatments. The values, shown in FIG. 5and expressed as per cent decrease in viability compared to theuntreated control cells, are the mean±std. dev. of 8 incubations in oneexperiment. Similar results were obtained in another experiment. Thedata show that during the observation period none of these compoundsexerted major toxic effects in the NIH 3T3 (normal) fibroblast cultures.In the MEL-28, A-549 and HT-29 cultures, CCDTHT was clearly moreeffective than CCcompound1 in reducing cell numbers. In the A-431 andMCF-7 cell lines, both agents exerted similarly large effects withCCDTHT being slightly more potent.

EXAMPLE 18 Long-Term Effects of CCDTHT on the Viability of Normal andCancer Cells

NIH3T3, T47D, A-431, HT-29, and MEL-28 cells were cultured as indicatedearlier. CaOV-3 human ovarian cancer cells were cultured in DMEMcontaining 10% FBS. ZR-75-1 human estrogen receptor-positive breastcancer cells were maintained in RPMI 1640 medium supplemented with 10%FBS. HTB-157 human fetal fibroblasts, MEL-24 human malignant melanomacells, Hep G2 human hepatoblastoma cells, and CaCO-2 human colonadenocarcinoma cells were maintained in Eagles's MEM containing 10% FBS.MB-231 human estrogen receptor-negative breast cancer cells weremaintained in Leibovitz's L-15 medium supplemented with 10% FBS. Cellswere seeded into 96-well plates and treated at ˜70-90% confluency. Twohours prior to treatments the medium was changed for fresh 10% serummedium, and then the cells were incubated for 10 days in the absence (□)or presence of 50 μM CCDTHT

or 100 μM CCDTHT (▪). The medium was changed and the cells werere-treated on days 3 and 6. The MTT assay was used to determine cellviability on day 10. The data, shown in FIG. 6, are the mean±std. dev.of 8 incubations in one experiment. (The experiment was repeated twicewith similar results). The results show that 100 μM CCDTHT causes 50% orless reduction in the number of viable cells in case of the two normalcell lines (HTB-157 and NIH 3T3 cells) and three cancer cell lines(MEL-24, HT-29, and CaCO-2). The T47D, ZR-75-1, CaOV-3, A-431 and MEL-28cells were very sensitive, while the MB-231 and HepG2 cells weremoderately sensitive to the inhibitory actions of CCDTHT. Clearly, 100μM CCDTHT does not universally induce strong reduction in the viabilityof all types of cancer cells.

EXAMPLE 19 Effects of CCDTHT on the Viability of Malignant Glioma Cellsand Astrocytomas

The U-87 human malignant glioma cells were cultured in MEM supplementedwith 10% FBS. The CCF-STTG1 human grade IV astrocytoma cells weremaintained in RPMI 1640 containing 1 mM glutamine and 10% FBS. Cellswere seeded into 12-well plates and treated at 60-70% confluency. Twohours prior to the treatments the medium was changed for fresh 10% serummedium, and then cells were incubated for 72 hours in the absence (□) orpresence of 50 μM CCDTHT

, 100 μM CCDTHT

, 150 μM CCDTHT

, or 200 μM CCDTHT (▪). The MTT assay was used to determine cellviability. The data, shown in FIG. 7, are the mean±std. dev. of 8incubations in one experiment (the experiment was repeated once withsimilar results). The results demonstrate that 100-200 μM concentrationsof CCDTHT sharply decrease the number of viable U-87 and CCF-STTG1cells. This suggests that CCDTHT may be particularly effective againstthe most dangerous forms of brain cancer.

EXAMPLE 20 Comparison of CCcompounds on the Viability of Normal MRC-5Lung Fibroblasts as well as T47D Breast Cancer and CaOV-3 Ovary CancerCells

The MRC-5 cell line is derived from normal lung tissue of a 14-week-oldmale fetus. MRC-5 cells were maintained and treated in Eagles Mediumwith Hanks' BSS containing 10% fetal bovine serum. The T47D cells weremaintained and treated in RPMI 1640 medium supplemented with 0.2 IUbovine insulin and 10% fetal bovine serum. The CaOV-3 human ovariancancer cells were cultured in Dulbecco's modified Eagle's mediumcontaining 10% fetal bovine serum. The cells were seeded in 96-wellplates either at 10,000 cells per well or 30,000 cells per well toexamine the antiproliferative and cytotoxic effects of various CCcompounds, respectively. The cells were incubated for 24 hours, then themedium was changed followed by the treatments of cultures 2 hours afterthe medium change. At this point, cell cultures seeded at 10,000 cellsper well and 30,000 cells per well were 25-35% and 80-90% confluent,respectively. Incubations were continued for 72 hours followed by theMTT assay. The data, shown in TABLE 2, indicate the inhibitoryconcentrations of CC compounds (IC₅₀) required for 50% decrease in thenumber of viable cells. These numbers are calculated from the mean of 4incubations in one experiment (the experiment was repeated once withsimilar results). The results indicate that while the ovary cancer cellsare the most sensitive to CCcompound24, in case of the T47D cancer cellsCCcompound26 had the greatest inhibitory effects. It is also clear thatwhile in case of CaOV-3 cells CCDTHT (CCcompound3) was more effectivethan CCcompound26, in case of T47D cells CCcompound26 was superior toCCDTHT. What is also important is that significantly greaterconcentrations of these CC compounds were required to inhibit theproliferation of normal fibroblasts (MRC-5 cells) than that of thesecancer cells. This suggests that at least in case of cancers of thebreast and ovary, CC compounds may be used to control tumor growthwithout significantly impacting the normal tissues.

TABLE 2 Comparison of CC compounds on the viability of normal MRC-5 lungfibroblasts as well as T47D breast cancer and CaOV-3 ovary cancer cells.IC₅₀ values Antiproliferative Cytotoxic CC compound MRC-5 T47D CaOV-3MRC-5 T47D CaOV-3 CCcompound3 90 47 38 119 66 39 CCcompound21 113 76 32164 59 29 CCcompound22 140 65 61 215 65 36 CCcompound23 139 48 14 163 3832 CCcompound24 113 56 69 207 59 29 CCcompound25 118 18 47 123 39 66CCcompound26 86 11 57 98 8 59

EXAMPLE 21 The Reversibility of CCDTHT Action on Cell Viability

HTB-157, MEL-28, CaOV-3, T47D, and A-431 cells were maintained asindicated earlier. The 3A-SubE post crisis placenta cells (capable ofunlimited proliferation) were maintained in Eagle's MEM containing 10%FBS. Cells were seeded into 96-well plates and treated at ˜70-90%confluency. Two hours before treatments the medium was changed for fresh10% serum medium, and then cells were incubated for 48 hours in theabsence (□) or presence of 50 μM CCDTHT

or 100 μM CCDTHT (▪). Half of the cultures was analyzed by the MTT assayat this point, and the other half of the cultures was incubated for anadditional 96 hours in fresh 10% serum-containing medium without CCDTHTfollowed by the MTT assay. The data, shown in FIG. 8, are the mean±std.dev. of 8 incubations in one experiment. (Similar results were obtainedin another experiment). HTB-157 (normal) cells, but none of the cancercell types, clearly resumed proliferation in fresh medium following thetreatment with 100 μM CCDTHT. In some cases (MEL-28 and 3A-SubE cells),pre-treatment with 50 μM CCDTHT was not sufficient to suppressproliferation. Overall, these results again indicate that a relativelyhigh dose of CCDTHT can irreversibly block proliferation of cancercells, while normal cells can resume proliferation more effectively.

EXAMPLE 22 Comparison of the Effects of Structurally Similar CCCompounds on the Proliferation of Normal and Cancer Cells

This experiment was performed to examine (i) how the melanoma cell linepairs (MEL-24 and MEL-28 cells), ovarian cancer cell line pairs (OVCARand CaOV-3 cells), and estrogen positive breast cancer cell line pairs(T47D and ZR-75-1), in each case two cell lines being derived fromsimilar tumors, respond to different CC compounds, and (ii) if any ofthe CC compounds are too toxic to normal cells (such as NIH 3T3 cells)which would preclude their use as anticancer agents. NIH 3T3, MEL-28,MEL-24, CaOV-3, ZR-75-1, and T47D cells were cultured as describedabove. The OVCAR human ovarian cancer cells were cultured in DMEMcontaining 10% FBS. Cells were seeded into 96-well plates and used at˜60-80% confluency. Two hours before treatments the medium was changedfor fresh 10% serum medium, and then the cells were incubated for 72hours in the absence or presence of 100 μM of each of CC compounds aslisted in TABLE 3. The inhibitory effects of CC compounds on cellproliferation are expressed as percent decrease compared to untreatedcell cultures (the greater the number, the greater the inhibitory effectof the CC compound). The data (average of 8 determinations) indicatethat CCcompound8 is too toxic to normal cells, precluding its use invivo. Also, for the treatment of ovarian cancer and breast cancer,CCDTHT seems to be the best choice (although in this experimentCCcompounds21-26 were not tested). In contrast, CCcompound12 orcombination of CCcompound12 with CCcompound11 or CCcompound14 appears tobe the most effective against melanoma. Similar comparative studies werealso performed with A-431 human epidermoid adenocarcinoma cells (CCDTHT,CCcompound9, and CCcompound12; 68-74% inhibition), PC-3 human prostatecancer cells (CCDTHT and CCcompound11; 54-55% inhibition), A549 humanlung adenocarcinoma cells (CCcompound11; 68% inhibition), MB-231 humanestrogen receptor-independent breast cancer cells (CCcompound11; 75%inhibition), and HT-29 human colon adenocarcinoma cells (CCcompound11and CCcompound14; 63-71% inhibition). The CC compounds indicated in theparentheses are the most effective inhibitors of proliferation of therespective cancer cell lines.

TABLE 3 Comparison of the effects of selected CC compounds (100 μM) onthe number of normal and cancer cells. Decrease in cell number (maximaldecrease = 100%) CC compound NIH 3T3 OVCAR CaOV-3 MEL-24 MEL-28 ZR-75-1T47D CCDTHT 29 63 84 15 60 74 61 CCcompound8 93 25 93 79 90 43 60CCcompound9 22 41 45 4 14 70 52 CCcompound10 21 38 45 15 11 65 51CCcompound11 21 62 66 20 94 75 59 CCcompound12 0 30 80 73 71 17 48CCcompound13 25 27 53 12 11 48 47 CCcompound14 30 65 69 21 90 53 58CCcompound15 11 26 28 0 19 41 35 CCcompound16 19 0 12 0 10 39 48

Examples comparing the effects of CCDTHT and the cholinetransport/choline kinase inhibitor hemicholinium-3 (HC-3) on cholinemetabolism and cell proliferation.

EXAMPLE 23 Comparison of Inhibitory Effects of Hemicholinium-3 (HC-3)and CCDTHT on Choline Metabolism in Cancer Cells

MCF-7 and MEL-28 cells were cultured as described earlier. The AN3CAhuman endometrial adenocarcinoma cells were maintained in Eagle's MEMcontaining 10% FBS. Each cell type was grown in 12-well plates toconfluency and then incubated with 1 μCi of [methyl-¹⁴C]choline for 2hours in the absence (□) or presence of 100 μM CCDTHT

or 0.5 mM HC-3 (▪), followed by the determination of cellular contentsof radiolabeled choline (A), PCho (B) and PtdCho (C), as indicated inFIG. 9. In each cell line, CCDTHT and HC-3 exerted comparably stronginhibitory effects on choline uptake and metabolism. Thus, if bothCCDTHT and HC-3 influenced cell viability solely via inhibition ofcholine metabolism, then these agents should similarly affect cellviability, at least at the concentrations used in the choline transportexperiment. It should be noted here, that at 0.1 mM concentration HC-3was a relatively weak inhibitor of choline transport (20-25% inhibition;data not shown here).

EXAMPLE 24 Comparison of Effects of CCDTHT and HC-3 on the Viability ofCancer Cells

AN3CA, HT-29, MEL-28, and MCF-7 cells were cultured as describedearlier. Cells were seeded into 96-well plates; when cells were ˜60-90%confluent, the medium was changed for fresh 10% serum-containing medium,followed by incubations for 72 hours in the absence (□) or presence of100 μM CCDTHT

or 0.5 mM HC-3 (▪). The MTT assay was used to determine cell viability.The data, shown in FIG. 10, are the mean±std. dev. of 8 incubations inone experiment. The results show that in AN3CA, HT-29, and MEL-28 cells,but not in MCF-7 cells, 100 μM CCDTHT causes much greater decreases inthe number of viable cells compared to 0.5 mM HC-3. The data areconsistent with the idea that in MCF-7 cells CCDTHT decreases cellviability purely via inhibiting choline metabolism, while in the othercell types CCDTHT acts by an additional mechanism probably eitherinvolving changes in the mitochondrial membrane and/or a presentlyunknown mechanism.

EXAMPLE 25 Ethanolamine Prevents the Effects of CCDTHT on Cell Viabilityonly in MCF-7 Cells

NIH 3T3, AN3CA, HT-29, MEL-28, and MCF-7 cells were cultured asdescribed earlier. Cells were seeded into 96-well plates; when cellswere ˜80-90% confluent, the medium was changed for fresh 10%serum-containing medium, followed by incubations for 72 hours in theabsence (□) or presence of 100 μM CCDTHT

, 2 mM ethanolamine (Etn)

, or 100 μM CCDTHT+2 mM Etn (▪). The MTT assay was used to determinecell viability. The data, shown in FIG. 11, are the mean±std. dev. of 8incubations in one experiment. In MCF-7 cells, but not in the othercancer cell lines, Etn clearly prevented the CCDTHT-induced reduction incell viability. While the reason for this is not entirely clear, thissuggests that CCDTHT inhibits cell proliferation by Etn-inhibited andnon-inhibited mechanisms, and that in MCF-7 cells CCDTHT acted mostlyvia the former mechanism(s).

Examples demonstrating the efficacy of CCDTHT in inhibitingproliferation of HaCaT keratinocytes.

EXAMPLE 26 CCDTHT Induces the Death of HaCaT Keratinocytes but Not HumanFetal Fibroblasts Treatment for 48 Hours

HaCaT keratinocytes, frequently used immortalized model cells forstudying psoriatic keratinocytes, and HTB-157 human fetus lungfibroblasts were cultured in 10% FBS-containing Dulbecco's modifiedEagle's medium (DMEM). Cells were distributed in 96-well plates; whencells were 50-60% confluent, the medium was changed for fresh 10%serum-containing medium, followed by incubations for 48 hours in theabsence (□) or presence of 50 μM CCDTHT

, or 100 μM CCDTHT (▪). The MTT assay was used to determine cellviability. The data, shown in FIG. 12, are the mean±std. dev. of 8incubations in one experiment (the experiment was repeated once withsimilar results). While in HaCaT cells, 50-100 μM CCDTHT strongly (by85-98%) reduced the number of viable cells, in HTB-157 fibroblasts thiscompound had much smaller effects (20-22% decrease in viability).Parallel microscopic examination of treated cells revealed that CCDTHTstrongly altered the morphology of HaCaT cells, consistent with celldeath, while CCDTHT had no visible effect on the morphology of HTB-157cells. Also, in HaCaT cells, 100 μM CCDTHT inhibited DNA synthesis byabout 95% after treatment for 48 hours, while a similar treatment causedonly about 16% decrease in DNA synthesis in the fibroblasts.Furthermore, when after treatment with CCDTHT the HaCaT cells werere-plated, no viable cells were found after incubations in fresh mediumfor 1 week. In contrast, there was practically no difference between thenumbers of re-plated untreated and treated (100 μM CCDTHT) HTB-157fibroblasts. This was a clear indication that the effects of CCDTHT onthe viability of HaCaT cells, unlike its smaller effects on theviability of normal fibroblasts, were irreversible.

EXAMPLE 27 Demonstration that After Treatment for 72 Hours CCDTHTPreferably Decreases the Viability of HaCaT Cells Compared toFibroblasts

HaCaT cells as well as HTB-157 and mouse embryo NIH 3T3 fibroblasts weresimilarly maintained in 10% FBS-containing DMEM, while 966 SK human skinfibroblasts were maintained in 10% serum-containing MEM. Cells weredistributed in 96-well plates; when cells were ˜60-80% confluent, themedium was changed for fresh 10% serum-containing medium, followed byincubations for 72 hours in the absence (□) or presence of 50 μM CCDTHT

, or 100 μM CCDTHT (▪). The MTT assay was used to determine cellviability. The data, shown in FIG. 13, are the mean±std. dev. of 8incubations in one experiment (the experiment was repeated once withsimilar results). Clearly, the data show that even after longertreatments, the three different fibroblast lines are significantly lesssensitive toward the inhibitory actions of CCDTHT than the HaCaT cells.

Comparison of effects of CCDTHT on four different cancer cell types invitro and after transplantation in vivo.

In these experiments the effects of CCDTHT were examined on theviability and growth of the same four tumor cell types in vitro as wellas in vivo after transplantation.

EXAMPLE 28 Comparison of the Effects of CCDTHT on the Viability ofVarious Types of Human Cancer Cells In Vitro

PC-3 human prostate adenocarcinoma cells were cultured in 12-well platesin Ham's F12 cell culture medium supplemented with 7% fetal bovineserum; the medium was changed for fresh 10% medium 2 hours prior totreatments. HL-60 human leukemia cells (in flasks) and HT 168 humanmelanoma cells were cultured in RPMI 1640 medium supplemented with 10%FCS; treatment of HL-60 and HT-168 cells were performed in flasks and12-well plates, respectively. T47D cells were cultured in 12-well platesin RPMI 1640 medium supplemented with 2 mM L-glutamine, 10 mM Hepes, 1mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 0.2l.U. bovine insulin/ml, and 10% fetal bovine serum; the medium waschanged for fresh 10% serum-containing medium 2 hours prior totreatments. All cells were incubated for 96 hours in the absence (□) orpresence of 25 μM CCDTHT

, 50 μM CCDTHT

, or 75 μM CCDTHT (▪). In this experiment, the viability of the cellcultures was estimated by Trypan blue exclusion using hematocytometer, atechnique well known to one having ordinary skill in the art. Threeindependently treated cell cultures, involving at least 300 cells foreach treatment, were analyzed. The experimental error is indicated asstd. dev. The results, shown in FIG. 14, indicate that the treatmentswith 50-75 μM concentrations of CCDTHT for four days strongly decreasedthe number of viable cancer cells; the HT-168, PC-3 and T47D cells werethe most sensitive to the actions of CCDTHT. The experiment with T47Dcells particularly well demonstrates that, in comparison to previousexperiments with the same cell lines, the effect of CCDTHT in vitro isdelayed but can be very powerful if sufficient time is allowed for itsaction to develop (i.e., 96 hours instead of 72 hours).

EXAMPLE 29 Effects of CCDTHT Alone on the Growth of Experimental HumanTumors

The experimental HL-60, HT-168, PC-3 and T47D human tumors weredeveloped in mice as described above. FIG. 15 depicts the effects of 50μmole

, 100 μmole

, 200 μmole

, and 400 μmole CCDTHT (▪), injected subcutaneously once daily, on thegrowth of these human tumor xenografts. “□” indicates the correspondingvalues in the untreated tumors. In a, b, c, and d, treatments started onday 18, 17, 12 and 17, respectively, and they were performed for 5+3days, 5+5 days, 5+3 days and 5+5 days, respectively, in each case with 2treatment-free days inserted between the 2 series of treatments.Treatments were terminated on day 29, 30, 23, and 30, respectively.Data, representing the average tumor size of 5 tumor-bearing mice foreach treatment, is expressed as % of the control tumor volume(untreated; 100%). The results indicate that 200-400 μmole amounts ofCCDTHT considerably reduced the growth of each tumor with the largestresponse being elicited in case of the HL-60 leukemia tumors. In none ofthe cases did CCDTHT cause significant weight loss, and the animalssurvived, on average, 3 to 8 days longer than without treatment.

Examples showing that CCDTHT is more effective in the presence ofpyrrolidinedithiocarbamate, zinc, and ethacrynic acid in reducing tumorvolume.

EXAMPLE 30 Effects of CCDTHT Alone and in Combination withPyrrolidinedithiocarbamate (PC)+Zinc (Zn) on the Growth of HT-168 HumanMelanoma Experimental Tumors

The HT-168 human melanoma tumors were developed as described above. Theywere untreated (), or treated once daily between 17-30 days on days 17,18, 19, 20, 21, 24, 25, 26, 27, and 28 with 200 μmole CCDTHT (▴), 100μmole PC+100 μmole Zn (▪), or 200 μmole CCDTHT+100 μmole PC+100 μmole Zn(♦). Each group consisted of 5 animals, and the mean values arepresented (the differences between the lowest and highest values werealways less than 11%). As shown in FIG. 16, the inhibitory effect ofCCDTHT on melanoma tumor growth was enhanced by PC+Zn in an additivemanner or even synergistically (at later time points). The mechanism ofcombined effect of PC and Zn is the subject of the U.S. Pat. No.6,756,063, titled “Methods and compositions for the treatment of humanand animal cancers”; issued on Jun. 29, 2004. In short, PC acts as acarrier for Zn through the cell membrane; i.e., PC is able to carry Zninside the cells which, if zinc achieves a certain intracellularconcentration, can lead to cell death. Considering that CCDTHT hadeffects on tumor size in several randomly selected tumors, it is areasonable expectation that CCDTHT in combination with PC and Zn willeffectively inhibit the growth of many different tumors. Based on thedifferential effects of various CC compounds on the viability andproliferation of different types of cancer cells, it is also reasonableto expect that different tumors will exhibit different sensitivitytoward CC compounds, so that CCDTHT may in some tumors be less effectivethan other CC compounds.

Examples showing that CCDTHT enhances the effects of Cisplatin (CisPt)on tumor volume while decreasing its toxic effects.

EXAMPLE 31 Combined Effects CCDTHT and CisPt on the Growth of MXT Tumoras well as Body Weight and Survival of the Experimental Animals

The mouse mammary MXT tumor was developed as described above. On day 7after transplantation of tumor cells, the animals were either remaineduntreated (group 1) or were treated with 2 mg/kg of CisPt alone (Group2), or 4 mg/kg of CisPt alone (Group 3), or 4.6 mg/kg of CCDTHT alone(Group 4), or 2 mg/kg of CisPt+4.6 mg/kg of CCDTHT (Group 5), or 4 mg/kgof CisPt+4.6 mg/kg of CCDTHT (Group 6). CCDTHT and CisPt wereadministered subcutaneously and intraperitoneally, respectively, oncedaily for 10 days, including a 2-day rest period after 5 days oftreatment (i.e., the last treatment was on day 19). Each group consistedof 7 animals, and the mean values±std. dev. for tumor volumes and bodyweight are shown in TABLE 4 and TABLE 5, respectively. While in thistumor model CCDTHT alone reduced the tumor volume less effectively thanin the human tumor models, it clearly added to the tumor volume-reducingeffect of CisPt (TABLE 4). A dramatic reduction in tumor volume wasparticularly evident in Group 6 (4 mg/kg CisPt+4.6 mg/kg CCDTHT). Thisexperiment indicated that CCDTHT may be used to enhance the effects ofchemotherapeutic agents on tumor volume.

As shown in TABLE 5, treatments with CisPt, particularly with the largerdose, caused significant decrease in body weight as a clear sign oftoxic effects on normal tissues. While CCDTHT alone had no effect onbody weight, it partially prevented CisPt-induced decrease in bodyweight at both concentrations of the latter.

TABLE 4 CCDTHT and CisPt in combination have greater effects on tumorvolume than separately in the MXT tumor model. Tumor volume (cm³) GroupTreatment Day 7 Day 14 Day 18 Day 21 1 None 0.44 ± 0.07 5.24 ± 0.27 8.61± 0.62 11.54 ± 1.36  2 CisPt, 2 mg/kg 0.46 ± 0.08 3.34 ± 1.04 5.37 ±0.68 6.23 ± 0.86 3 CisPt, 4 mg/kg 0.45 ± 0.10 1.77 ± 0.40 all dead alldead 4 CCDTHT, 4.6 mg/kg 0.43 ± 0.07 4.14 ± 0.71 6.62 ± 0.43 9.35 ± 0.695 CisPt, 2 mg/kg + 0.48 ± 0.12 2.88 ± 0.47 4.69 ± 0.79 5.32 ± 0.46CCDTHT, 4.6 mg/kg 6 CisPt, 4 mg.kg + 0.43 ± 0.15 1.64 ± 0.37 2.51 ± 0.353.76 ± 0.31 CCDTHT, 4.6 mg/kg

TABLE 5 CCDTHT partially prevents CisPt-induced reduction in body weightin the MXT tumor model. Body weight (g) Group Treatment Day 7 Day 14 Day18 Day 21 1 None 22.9 ± 0.89 25.6 ± 1.07 27.3 ± 0.70 29.6 ± 1.52 2CisPt, 2 mg/kg 22.4 ± 0.73 22.9 ± 0.87 22.3 ± 1.02 21.2 ± 1.31 3 CisPt,4 mg/kg 22.5 ± 0.92 19.4 ± 1.05 all dead all dead 4 CCDTHT, 4.6 mg/kg22.8 ± 0.82 25.1 ± 0.84 26.9 ± 0.93 28.8 ± 0.91 5 CisPt, 2 mg/kg + 22.5± 0.95 22.6 ± 0.53 24.2 ± 0.90 27.0 ± 1.51 CCDTHT, 4.6 mg/kg 6 CisPt, 4mg.kg + 22.3 ± 1.06 22.3 ± 1.50 23.6 ± 1.59 25.9 ± 1.30 CCDTHT, 4.6mg/kg

On average, control animals survived for 26.8±2.4 days, while animals ingroups 2, 3, 4, 5, and 6 survived for 32.7±6.2, 18.1±0.7, 30.6±3.2,34.7±5.4 and 25.1±3.3 days, respectively. The most dramatic effect ofCCDTHT was its ability to enhance the survival of 4 mg/kg CisPt-treatedgroup from 18 to 25 days. It clearly indicates that the simultaneous useof CCDTHT allows the use of higher, more effective doses of CisPt, andby implication of other chemotherapeutic agents. It should be noted thatmost animals with large treated tumors died because of various lungproblems. Since in humans there are effective methods to protect thelung's functions, it is reasonable to expect that the effect of CCDTHTon the survival of CisPt-treated human patients will prove to be evengreater.

Examples showing that placental alkaline phosphatase (PALP) alone or incombination with CisPt exhibits anticancer effects.

EXAMPLE 32 Combined Effects of PALP and CisPt on the Growth of MXT Tumoras well as Body Weight and Survival of the Experimental Animals

The mouse mammary MXT tumor model was developed and treated as describedabove. On day 7 the animals were either remained untreated (Group 1) orwere treated with 3 mg/kg of CisPt (Group 2) or 3 mg/kg of CisPt+15mg/kg of highly purified PALP (Group 3). CisPt and PALP wereadministered intraperitoneally and subcutaneously, respectively, seventimes on every second day (i.e., treatments were terminated on day 21).Each group included 7 animals; the mean values±std. dev. for tumorvolumes and body weight are shown in TABLE 6 and TABLE 7, respectively.The data show that on each day examined, PALP consistently enhanced theinhibitory effects of CisPt on tumor growth (TABLE 6). In addition, PALPfully restored body weight lost as a consequence of CisPt treatment(TABLE 7).

Probably as a combination of effects of PALP on tumor volume, PALP alsoenhanced the survival of animals. While the control and CisPt-treatedanimals survived for 24.2±2.2 and 31.1±6.2 days, respectively, theCisPt+PALP-treated animals survived for 37.7±3.68 days. Based on theseresults, it is reasonable to expect that PALP will enhance the efficacyof other cancer treatments as well with parallel extension of survivaltime and reduction of body weight loss.

TABLE 6 PALP enhances the inhibitory effects of CisPt on the growth ofMXT tumors. Tumor volume (cm³) Group Treatment Day 7 Day 11 Day 16 Day21 1 None 0.35 ± 0.09 2.94 ± 0.75 7.98 ± 1.25 11.13 ± 2.70  2 CisPt 0.38± 0.01 1.45 ± 0.22 4.53 ± 1.00 5.86 ± 1.38 3 CisPt + PALP 0.33 ± 0.061.16 ± 0.24 3.32 ± 0.87 4.28 ± 0.97

TABLE 7 PALP prevents CisPt-induced body weight loss in the MXT tumormodel. Body weight (g) Group Treatment Day 7 Day 11 Day 16 Day 21 1 None22.3 ± 0.61 24.5 ± 0.91 25.9 ± 1.13 27.7 ± 1.34 2 CisPt 22.7 ± 0.37 23.1± 0.52 20.9 ± 0.65 19.2 ± 0.66 3 CisPt + PALP 22.1 ± 0.52 24.2 ± 0.2725.6 ± 0.26 27.1 ± 0.49

Examples showing that CCDTHT in combination with PALP has greateranticancer effects than alone.

EXAMPLE 33 CCDTHT in Combination with PALP More Effectively Inhibits theGrowth of Leukemia Tumor and Promotes Survival than Alone

The HL-60 human leukemia model was developed and treated as describedabove. On day 12 the mice either remained untreated (Group 1) or weretreated with 4.6 mg/kg CCDTHT (Group 2) or 4.6 mg/kg CCDTHT+15 mg/kghighly purified PALP (Group 3). Both CCDTHT and PALP were administeredsubcutaneously for three 5-day cycles with 2 resting days includedbetween each 5-day cycle. In each group 7 mice were included. The meanvalues±std. dev. for tumor volumes are shown in TABLE 8. The data showthat CCDTHT inhibits the growth of leukemia tumor and that CCDTHT+PALPhave greater inhibitory effects than CCDTHT alone.

The two agents had even more pronounced combined effects on the survivalof leukemic mice. While the untreated mice survived for 28.9±3.4 days,mice treated with CCDTHT alone and CCDTHT+PALP survived for 34.9±6.6 and46.3±9.7 days, respectively. The remarkable 60% increase in survival byCCDTHT+PALP is a strong indication that human leukemia may be one ofthose cancers where the CCDTHT/PALP combination, probably in combinationwith other therapies, can be used successfully to control cancer cellgrowth and enhance survival.

TABLE 8 Combined inhibitory effects of CCDTHT and PALP on the growth ofhuman leukemia xenografts. Tumor volume (cm³) Group Treatment Day 12 Day16 Day 22 Day 26 1 None 0.43 ± 0.21 1.55 ± 0.62 3.87 ± 0.59 5.73 ± 0.752 CCDTHT 0.46 ± 0.32 1.26 ± 0.73 2.74 ± 0.37 3.72 ± 0.44 3 CCDTHT + PALP0.48 ± 0.20 0.88 ± 0.27 2.14 ± 0.72 3.07 ± 0.53

EXAMPLE 34 CCDTHT and PALP in Combination Restore Body Weight andEnhance the Inhibitory Effects of CisPt on the Growth of MXT Tumors

The mouse mammary MXT tumor model was developed and treated as describedabove. On day 7 the animals either remained untreated (Group 1) or weretreated with 3 mg/kg CisPt (Group 2), 3 mg/kg CisPt+4.6 mg/kg of CCDTHT(Group 3), or 3 mg/kg CisPt+4.6 mg/kg of CCDTHT+15 mg/kg highly purifiedPALP (Group 4). CisPt was administered intraperitoneally 9-times onceevery second day; CCDTHT was administered for 15 days once daily; andPALP was administered 3 times every 3^(rd) day. In each group 7 micewere included. The mean values±std. dev. for tumor volumes and bodyweights are shown in TABLE 9 and TABLE 10, respectively.

The results show that CCDTHT and PALP in combination significantlyenhance the inhibitory effects of CisPt on the growth of MXT tumors(TABLE 9). In addition, CCDTHT alone, and particularly in combinationwith PALP, prevents the serious loss of body weight induced by CisPt(TABLE 10). Finally, the two agents together increased the survival timefrom 34 days, observed with CisPt alone, to 40 days.

TABLE 9 CCDTHT and PALP in combination enhance the inhibitory effects ofCisPt on the growth of MXT tumors. Tumor volume (cm³) Group TreatmentDay 7 Day 11 Day 15 Day 21 1 None 0.38 ± 0.13 2.21 ± 0.47 5.98 ± 0.7612.66 ± 2.09  2 CisPt 0.36 ± 0.12 1.92 ± 0.67 3.80 ± 1.30 6.64 ± 1.28 3CisPt + CCDTHT 0.40 ± 0.10 1.63 ± 0.24 3.16 ± 0.39 5.14 ± 0.71 4 CisPt +0.41 ± 0.07 1.03 ± 0.12 2.40 ± 0.32 3.72 ± 0.53 CCDTHT + PALP

TABLE 10 CCDTHT and PALP in combination prevent CisPt- induced loss ofbody weight. Body weight (g) Group Treatment Day 7 Day 11 Day 15 Day 211 None 22.4 ± 0.60 24.06 ± 0.56 25.6 ± 0.65 27.4 ± 0.70 2 CisPt 22.2 ±1.10 23.10 ± 1.40 22.9 ± 1.00 20.6 ± 1.80 3 CisPt + CCDTHT 22.5 ± 0.79 23.8 ± 0.54 24.1 ± 0.94 26.7 ± 0.97 4 CisPt + CCDTHT + 22.0 ± 0.38 23.6 ± 0.79 25.4 ± 1.09 27.9 ± 0.75 PALP

1. A composition administered to a patient with cancer attherapeutically effective doses to reduce tumor size in order to preventtumor-induced or treatment-induced patient weight loss, which increasespatient survival, wherein the composition comprises alkaline phosphataseor a physiologically active derivative thereof wherein the derivative isselected from fragments of alkaline phosphatase that demonstrateefficiencies similar or more effective than native alkaline phosphatase,a suitable carrier, and a heterocyclic compound having one or twoquaternary ammonium groups represented by the formula:

wherein R₁ and R₃₋₈ are independently hydrogen, C₁-C₂₆ straight,branched or cyclic alkanes or alkenes, aromatic hydrocarbons, alcohols,ethers, aldehydes, ketones, carboxylic acids, amines, amides, nitriles,or five- and/or six-membered heterocyclic moieties; wherein R₉ and R₁₀considered together are ═O or ═CH-L-N⁺(R₁₁, R₁₂, R₁₃) or wherein R₉ andR₁₀ considered independently are —OH or -L-N⁺(R₁₁, R₁₂, R₁₃); wherein R₂is hydrogen, —CH₃, —X—L-N+(R₁₁, R₁₂, R₁₃)Z⁻; or -L-N⁺(R₁₁, R₁₂, R₁₃)Z⁻;wherein V is —S⁻, —Se⁻, —C⁻, —O⁻ or —N; wherein Y is —S⁻, —Se⁻, —C⁻, —O⁻or —N; wherein —X is —CH₂ ⁻, —OCH₂ ⁻, —CH2O⁻, SCH2⁻ or —CH₂S⁻; wherein Lis a C₁-C₄ straight alkane, alkene, thiol, ether, or amine; wherein R₁₁,R₁₂ and R₁₃ are independently C₁-C₄ straight alkanes, alkenes, thiols,amines, ethers or alcohols; and wherein Z⁻ is Cl⁻, Br⁻ or I⁻.
 2. Thecomposition of claim 1 wherein R₁₁, R₁₂, and R₁₃ are independentlymethyl, ethyl, propyl, allyl, ether, sulfhydryl, amino, or hydroxylgroups.
 3. The composition of claim 1 wherein the heterocyclic compoundis a thioxanthone and R₂ is -X-L-N⁺(R₁₁, R₁₂, R₁₃)Z⁻.
 4. The compositionof claim 1 wherein the heterocyclic compound is[3-(3,4-dimethyl-9-oxo-9H-thioxanthen-2-yloxy)-2-hydroxypropyl]trimethylammonium chloride.
 5. The composition of claim 1 wherein theheterocyclic compound isN,N,-diethyl-N-methyl-2-[9-oxo-9H-thioxanthen-2-yl)methoxy] ethanaminiumiodide.
 6. The composition of claim 1 wherein the heterocyclic compoundis a thioxanthene and wherein R₂ hydrogen or CH₃, R₉ and R₁₀ consideredtogether are ═CH-L-N⁺(R₁₁, R₁₂, R₁₃); L is —(CH₂)₂— or —(CH₂)₃—; and R₁and R₃₋₈ are hydrogen.
 7. The composition of claim 6 wherein theheterocyclic compound isN,N-diethyl-N-allyl-3-(2-methyl-9H-thioxanthen-9-ylidene)-propane-1-aminiumbromide.
 8. The composition of claim 6 wherein the heterocyclic compoundisN,N,N-trimethyl-3-(2-methyl-9H-thioxanthen-9-ylidene)-propane-1-aminiumiodide.
 9. The composition of claim 6 wherein the heterocyclic compoundis N,N,N-trimethyl-3-(9H-thioxanthen-9-ylidene)-propane-1-aminiumiodide.
 10. The composition of claim 1, wherein the R₁₁, R₁₂, or R₁₃ inthe heterocyclic trialkylammonium compound is modified by a targetingmoiety comprising antibody, folic acid, steroid, fatty acid or a peptidedeveloped to bind to a specific cell surface protein or cross the cellmembrane.
 11. The composition of claim 1 wherein the alkalinephosphatase is obtained from mammalian tissue.
 12. The composition ofclaim 11 wherein the alkaline phosphatase is placental alkalinephosphatase, intestinal alkaline phosphatase, tissue non-specificalkaline phosphatase or placental alkaline phosphatase-like germ-celltype alkaline phosphatase.
 13. The composition of claim 11 wherein thealkaline phosphatase is a human alkaline phosphatase.
 14. Thecomposition of claim 1 wherein the alkaline phosphatase is produced by arecombinant method with a corresponding coding gene sequence beingtransferred into and expressed in a single cell or multi cell organism.15. The composition of claim 1 wherein the composition is administeredby an oral, topical, intravenous, intraarterial, subcutaneous,intraperitoneal, intradermal, intratissue, intramuscular, intraportal,intracranial, infusion, or aerosol delivery route, or by a minipump. 16.The composition of claim 1 wherein the alkaline phosphatase andheterocyclic compound are administered in a suitable carriersimultaneously.
 17. The composition of claim 1 wherein the alkalinephosphatase and heterocyclic trialkylammonium compound components areadministered in a suitable carrier separately once daily, or once,twice, or three times a week.
 18. The composition of claim 1 wherein thealkaline phosphatase and heterocyclic trialkylammonium compoundcomponents are administered in a suitable carrier simultaneously orseparately via different administration routes.
 19. The composition ofclaim 1 wherein a suitable carrier is 0.9% sodium chloride and theadministration is via an injection method.
 20. The composition of claim1 wherein the composition suitable carrier is a vaseline-based materialand administration is via a topical administration method.
 21. Thecomposition of claim 1 wherein the composition is a tablet, gel capsule,or a liquid composed of one or more ingredients conforming to industrialstandards, and administrated orally.